Method for producing ethanol and co-products from cellulosic biomass

ABSTRACT

The present invention generally relates to processes for production of ethanol from cellulosic biomass. The present invention also relates to production of various co-products of preparation of ethanol from cellulosic biomass. The present invention further relates to improvements in one or more aspects of preparation of ethanol from cellulosic biomass including, for example, improved methods for cleaning biomass feedstocks, improved acid impregnation, and improved steam treatment, or “steam explosion.”

REFERENCE TO RELATED APPLICATIONS

This application is a United States National Stage Application based onInternational Patent Application No. PCT/US2010/046561, filed Aug. 24,2010, and claims the benefit of U.S. Provisional Patent Application Ser.No. 61/236,345, filed Aug. 24, 2009, the entire contents of which areincorporated herein by reference.

GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under DOE CooperativeAgreement Nos. DE-FC36-03GO13142 and DE-FC36-07GO17028. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to processes for production ofethanol from cellulosic biomass. The present invention also relates toproduction of various co-products of preparation of ethanol fromcellulosic biomass. The present invention further relates toimprovements in one or more aspects of preparation of ethanol fromcellulosic biomass including, for example, improved methods for cleaningbiomass feedstocks, improved acid impregnation, and improved steamtreatment, or “steam explosion.”

BACKGROUND OF THE INVENTION

Lignocellulosic biomass is a complex structure comprising cellulose,hemicellulose, and lignin in which cellulose and hemicellulose are boundto the lignin. Cellulose is a polymer of D-glucose with β [1-4] linkagesbetween each of the about 500 to 10,000 glucose units. Hemicellulose isa polymer of sugars, primarily D-xylose with other pentoses and somehexoses with β [1-4] linkages derived from herbaceous materials andvarious hardwood species. Lignin is a complex random polyphenolicpolymer.

There are a variety of widely available sources of lignocellulosicbiomass including, for example, corn stover, agricultural residues(e.g., straw, corn cobs, etc.), woody materials, energy crops (e.g.,sorghum, poplar, etc.), and bagasse (e.g., sugarcane). Thus,lignocellulosic biomass is a relatively inexpensive and readilyavailable substrate for the preparation of sugars, which may befermented to produce alcohols such as ethanol. Ethanol has a number ofuses, including in fuel. For example, ethanol may be used as an additiveto gasoline to boost octane, reduce pollution, and/or to partiallyreplace gasoline and reduce crude oil requirements.

Generally, preparation of ethanol from lignocellulosic biomass involves(1) liberating cellulose and hemicellulose from lignin and/or increasingthe accessibility of cellulose and hemicellulose to enzymatichydrolysis, (2) depolymerizing carbohydrate sugars of hemicellulose andcellulose to free sugars, and (3) fermenting the sugars to ethanol.

Processes for preparation of ethanol from lignocellulosic biomass areknown, but there remains an unfulfilled need for an ethanol productionprocess that may be practiced economically on a commercial scale. Forexample, the need exists for ethanol production processes that provideimproved ethanol yields over conventional processes and/or provideuseful, improved co-products of ethanol production.

SUMMARY OF THE INVENTION

Briefly, therefore, the present invention is directed to improvedprocesses for production of ethanol that provide one or moreadvantageous results including, for example, improved ethanol yieldand/or improved co-products of ethanol production.

The present invention is also directed to methods for cleaning a biomassfeedstock. In one embodiment, the method comprises removing from thebiomass feedstock a fine particulate fraction, wherein the fineparticulate fraction has a particle size distribution such that at leastabout 95 wt % of the particles pass through a screen having openings ofa size of about U.S. Sieve No. 20 (840 μm), thereby forming a cleanedbiomass feedstock having an ash content of no more than about 75% of theash content of the biomass feedstock (dry weight basis).

In another embodiment, the method comprises removing from the biomassfeedstock a fine particulate fraction, wherein the fine particulatefraction has a particle size distribution such that at least about 95 wt% of the particles pass through a screen having openings of a size ofabout U.S. Sieve No. 20 (840 μm), thereby forming a cleaned biomassfeedstock having an ash content of less than about 8 wt % (dry weightbasis).

In another embodiment, the method comprises removing from the biomassfeedstock a fine particulate fraction, thereby forming a cleaned biomassfeedstock, wherein the fine particulate fraction has a particle sizedistribution such that at least about 95 wt % of the particles passthrough a screen having openings of a size of about U.S. Sieve No. 20(840 μm), the ash content of the cleaned biomass feedstock comprises anacid soluble fraction and an acid insoluble fraction, and the acidsoluble ash fraction constitutes at least about 30 wt % of the ashcontent of the cleaned biomass feedstock.

In a further embodiment, the method comprises removing from the biomassfeedstock a fine particulate fraction comprising ash, thereby forming acleaned biomass feedstock, wherein the ratio of ash content of the fineparticulate fraction to the ash content of the biomass feedstock is atleast about 3:1.

In a still further embodiment, the method comprises removing from thebiomass feedstock a fine particulate fraction, thereby forming a cleanedbiomass feedstock, wherein the ratio of the ash content of the fineparticulate fraction to the ash content of the cleaned biomass feedstockis at least about 5:1.

The present invention is also directed to methods for cleaning acellulosic biomass feedstock comprising corn stover having an ashcontent of at least 3 wt %. In one embodiment, the method comprisesremoving from the biomass feedstock a fine particulate fraction, whereinthe fine particulate fraction has a particle size distribution such thatat least about 95 wt % of the particles pass through a screen havingopenings of a size of about U.S. Sieve No. 20 (840 μm), thereby forminga cleaned biomass feedstock having an ash content of no more than about75% of the ash content of the biomass feedstock (dry weight basis). Inanother embodiment, the method comprises removing from the biomassfeedstock a fine particulate fraction comprising ash, thereby forming acleaned biomass feedstock, wherein the ratio of ash content of the fineparticulate fraction to the ash content of the biomass feedstock is atleast about 3:1.

The present invention is further directed to methods for cleaning acellulosic biomass feedstock wheat straw having an ash content of atleast 3 wt %. In one embodiment, the method comprises removing from thebiomass feedstock a fine particulate fraction, wherein the fineparticulate fraction has a particle size distribution such that at leastabout 95 wt % of the particles pass through a screen having openings ofa size of about U.S. Sieve No. 20 (840 μm), thereby forming a cleanedbiomass feedstock having an ash content of no more than about 75% of theash content of the biomass feedstock (dry weight basis). In anotherembodiment, the method comprises removing from the biomass feedstock afine particulate fraction comprising ash, thereby forming a cleanedbiomass feedstock, wherein the ratio of ash content of the fineparticulate fraction to the ash content of the biomass feedstock is atleast about 3:1.

The present invention is further directed to methods for pretreatment ofcellulosic biomass feedstock comprising cellulose, hemicellulose, andlignin.

The present invention is also directed to methods for pretreatment ofparticulate cellulosic biomass feedstock that comprise removing a fineparticulate fraction from the biomass feedstock. In one embodiment, acleaned particulate biomass feedstock having an acid neutralizationcapacity as determined in accordance with Protocol A of less than 0.01is formed. In another embodiment, a cleaned particulate biomassfeedstock having an acid neutralization capacity as determined inaccordance with Protocol A that is no more than about 90% of the acidneutralization capacity of the biomass feedstock is formed.

In one embodiment, the method comprises removing from the particulatebiomass feedstock a fine particulate fraction, thereby forming a cleanedparticulate biomass feedstock; contacting the cleaned particulatebiomass feedstock with an acidic liquid medium in an acid impregnationzone to form an acid-impregnated cellulosic biomass feedstock, theweight ratio of acid to solids fraction of the cleaned particulatebiomass feedstock introduced into the acid impregnation zone is lessthan about 0.05:1; and contacting the acid-impregnated biomass feedstockwith water at elevated temperature and pressure in a pretreatment zone,thereby forming a pretreated biomass feedstock comprising a solidsfraction and a liquid fraction comprising xylose, wherein the xylosecontent, as determined in accordance with Protocol B, of the pretreatedbiomass feedstock liquid fraction represents a yield of at least about70% (based on hemicellulose content of the particulate biomassfeedstock).

In another embodiment, the method comprises removing from theparticulate biomass feedstock a fine particulate fraction, therebyforming a cleaned particulate biomass feedstock; contacting the cleanedparticulate biomass feedstock with an acidic liquid medium in an acidimpregnation zone to form an acid-impregnated cellulosic biomassfeedstock, the weight ratio of acid to solids fraction of the cleanedparticulate biomass feedstock introduced into the acid impregnation zoneis less than about 0.05:1; and contacting the acid-impregnated biomassfeedstock with water at elevated temperature and pressure in apretreatment zone, thereby forming a pretreated biomass feedstockcomprising a solids fraction comprising cellulose; the cellulosedigestibility of the pretreated biomass feedstock as determined inaccordance with Protocol C is at least about 60%.

In one embodiment, the method comprises contacting the cellulosicbiomass feedstock with an acidic liquid medium to form anacid-impregnated biomass feedstock; contacting the acid-impregnatedcellulosic biomass feedstock with H₂O at elevated temperature andpressure within a contact zone under conditions effective forsolubilizing hemicellulose and producing a steam treated feedstock;subjecting the steam treated feedstock within a depressurization zone toconditions effective for solubilizing hemicellulose and producing avolatilized fraction of the steam treated feedstock; and releasing atleast a portion of the volatilized fraction from the depressurizationzone for control of temperature and pressure within the depressurizationzone, wherein control of the temperature and pressure within thedepressurization zone consists essentially of releasing at least aportion of the volatilized fraction therefrom.

The present invention is also directed to methods for pretreatment ofvirgin cellulosic biomass feedstock comprising cellulose, hemicellulose,and lignin. In one embodiment, the method comprises contacting thecellulosic biomass feedstock and an acidic aqueous liquid medium to forman acid-impregnated cellulosic biomass feedstock containing less than 50wt % aqueous liquid on a water basis.

The present invention is further directed to methods for pretreatment ofparticulate cellulosic biomass feedstock comprising cellulose,hemicellulose, and lignin. In one embodiment the method comprisescontacting the cellulosic biomass feedstock and an acidic aqueous liquidmedium to form an acid-impregnated cellulosic biomass feedstock, whereinat least about 50 wt % of the feedstock particles have a size in theirlargest dimension of from about 0.6 cm (0.25 inches) to about 4 cm (1.5inches).

In one embodiment, the method comprises spraying an acidic liquid mediumonto the cellulosic biomass feedstock to form an acid-impregnatedcellulosic biomass feedstock; and contacting the acid-impregnatedcellulosic biomass feedstock with H₂O for between about 1 and about 120minutes at elevated temperature within a contact zone containing a vaporphase wherein the partial pressure of water vapor is at least about 55psig.

In another embodiment, the method comprises spraying an acidic liquidmedium onto the cellulosic biomass feedstock to form an acid-impregnatedcellulosic biomass feedstock, and agitating the feedstock to distributethe medium within the feedstock and bring particles of the feedstockinto mutually abrading contact.

In another embodiment, the method comprises spraying an acidic liquidmedium onto the cellulosic biomass feedstock to form an acid-impregnatedcellulosic biomass feedstock in a contact zone, wherein the contact zonecomprises parallel counter-rotating shafts having flights mountedthereon for agitation of the biomass.

In a still further embodiment, the method comprises, contacting thecellulosic biomass feedstock with an aqueous liquid medium comprising anacid and a surfactant (wetting agent) to form an acid-impregnatedbiomass feedstock.

In another embodiment, the method comprises contacting the cellulosicbiomass feedstock and an acidic liquid medium to form anacid-impregnated cellulosic biomass feedstock; contacting theacid-impregnated cellulosic biomass feedstock with H₂O at elevatedtemperature within a contact zone containing a vapor phase wherein thepartial pressure of water vapor is at least about 55 psig to solubilizehemicellulose and produce a volatilized fraction of the acid-impregnatedfeedstock; and releasing at least a portion of the volatilized fractionfrom the contact zone at a rate effective to control the pressure in thecontact zone.

In another embodiment, the method comprises introducing the feedstockinto a steam contact zone, the contact zone having an inlet for steamand an outlet for pretreated feedstock; introducing steam into thecontact zone at the inlet to contact steam and the feedstock and form asteam treated feedstock; and removing pretreated feedstock from thecontact zone through the outlet and into a receiving zone, wherein thepressure in the receiving zone does not differ from the pressure in thecontact zone by more than about 200 psig.

In another embodiment, the method comprises introducing the feedstockinto a steam contact zone; introducing steam into the contact zone tocontact the feedstock and form a steam-treated feedstock; and passingthe steam-treated feedstock from the steam contact zone through a flowrestriction and into a receiving zone, the pressure drop across the flowrestriction being less than about 150 psi.

In another embodiment, the method comprises contacting the cellulosicbiomass feedstock with H₂O within a contact zone containing a vaporphase wherein the partial pressure of water vapor is at least about 55psig, the H₂O being distributed within the zone so that the biomass isbrought to a target temperature, and the average temperature of anyregion of the biomass that contains more than 15% by weight of thebiomass does not differ by more than 5° C. from the target temperature.

The present invention is further directed to methods for pretreatment ofcellulosic biomass feedstock comprising cellulose, hemicellulose,lignin, and one or more impurities. In one embodiment, the methodcomprises contacting the cellulosic biomass feedstock with an acidicaqueous liquid medium to form an acid-impregnated cellulosic biomassfeedstock; and removing an aqueous liquid fraction from theacid-impregnated cellulosic biomass feedstock to form anacid-impregnated feedstock having a reduced content of the one or moreimpurities.

The present invention is further directed to methods for washing avirgin solid phase biomass feedstock comprising cellulose,hemicellulose, and lignin. In one embodiment, the method comprisescontacting the cellulosic biomass with an aqueous washing liquid andthereafter separating the resulting wash liquor from the solid phasebiomass, the biomass being contacted with the washing liquid underconditions that do not degrade the fibers by more than 20% as measuredby the average length of fibers in the biomass after the contacting ascompared to the average length of fibers in the biomass before thecontacting.

The present invention is further directed to methods for recovering C₅sugars from cellulosic biomass feedstock comprising cellulose,hemicellulose and lignin. In one embodiment, the method comprisespretreating the biomass feedstock in the presence of an aqueous liquidmedium; contacting the pretreated feedstock with a hemicellulase toproduce a hydrolyzate slurry comprising an aqueous phase containing C₅sugar(s) and a solid phase comprising cellulose and lignin; andseparating an aqueous liquid hydrolyzate fraction comprising C₅ sugar(s)from the hydrolyzate slurry.

The present invention is further directed to methods for producingfermentable sugars from a cellulosic biomass feedstock comprisingcellulose, hemicellulose, and lignin. In one embodiment, the methodcomprises contacting the cellulosic biomass feedstock with an acidicliquid medium to form an acid-impregnated cellulosic biomass feedstock;forming a pretreated cellulosic biomass feedstock, the formingcomprising contacting the acid-impregnated cellulosic biomass feedstockwith H₂O at elevated temperature and pressure; contacting the pretreatedcellulosic biomass feedstock with a hemicellulase enzyme to hydrolyzehemicellulose and produce hemicellulose-derived fermentable sugars in ahemicellulose hydrolyzate comprising a liquid phase comprisingsolubilized hemicellulose-derived fermentable sugars and a solid phasecomprising cellulose and lignin; and removing an aqueous liquid phasecomprising hemicellulose-derived fermentable sugars from the pretreatedhydrolyzate.

The present invention is further directed to methods for conversion ofcellulose to glucose in an aqueous hydrolysis medium. In one embodiment,the method comprises contacting glucose, cellulose, a nitrogen source,and a microbe that is effective to express a cellulase enzyme in anaqueous biosynthesis medium within a microbe proliferation zone therebyproducing cellulase enzyme within the proliferation zone; transferringcellulase from the proliferation zone to a cellulose hydrolysis zonewherein cellulase is contacted with cellulose in a cellulase hydrolysismedium; and enzymatically hydrolyzing cellulose in the cellulasehydrolysis medium within the enzymatic hydrolysis zone, therebygenerating C₆ sugars.

The present invention is further directed to methods for producing acellulase enzyme from virgin cellulosic biomass feedstock comprisingcellulose, hemicellulose, and lignin. In one embodiment, the methodcomprises contacting the cellulosic biomass feedstock with an acidicliquid medium to form an acid-impregnated cellulosic biomass feedstock;forming a pretreated cellulosic biomass feedstock, the formingcomprising contacting the acid-impregnated cellulosic biomass feedstockwith H₂O at elevated temperature and pressure; hydrolyzing hemicelluloseof the pretreated cellulosic biomass feedstock to producehemicellulose-derived fermentable sugars in a hemicellulose hydrolyzatecomprising a liquid phase comprising solubilized hemicellulose-derivedfermentable sugars and a solid phase comprising cellulose and lignin;separating an aqueous liquid hydrolyzate fraction comprisinghemicellulose-derived fermentable sugars from the pretreatedhydrolyzate; and contacting in a proliferation zone a portion of thesolid phase comprising cellulose, a nitrogen source, and a microbe thatis effective to express a cellulase enzyme, thereby producing cellulaseenzyme within the proliferation zone.

The present invention is further directed to methods for producingand/or recovering ethanol from a cellulosic biomass feedstock comprisingcellulose, hemicellulose, and lignin.

In one embodiment, the method comprises pretreating the biomass toincrease the bioavailability of the hemicellulose and cellulosecontained therein; contacting the pretreated biomass with ahemicellulase to cause hemicellulose to be hydrolyzed to yield solubleC₅ sugar(s) and produce a hemicellulase hydrolyzate slurry comprising anaqueous phase containing C₅ sugar(s) and a solid phase comprisingcellulose and lignin; separating an aqueous phase C₅ fraction comprisingC₅ sugar(s) from the hemicellulase hydrolyzate slurry, yielding athickened residual fraction comprising a cake or concentrated slurrycomprising the solid phase cellulose and lignin; contacting C₅ sugarsobtained in the aqueous phase C₅ fraction with a yeast, therebyconverting C₅ sugar(s) to ethanol and producing a C₅ fermentatecontaining ethanol; contacting cellulose of the thickened fraction witha cellulase, thereby converting cellulose to C₆ sugar(s) and producing aC₆ hydrolyzate; and contacting C₆ sugars produced in the C₆ hydrolyzatefraction with a yeast, thereby converting C₆ sugar(s) to ethanol andproducing a C₆ fermentate containing ethanol.

In another embodiment, the method comprises contacting the cellulosicbiomass feedstock with an acidic aqueous liquid medium to form anacid-impregnated cellulosic biomass feedstock; forming a pretreatedfeedstock comprising solubilized hemicellulose and a solid phasecomprising cellulose and lignin, the forming comprising contacting theacid-impregnated cellulosic biomass feedstock with H₂O at elevatedtemperature and pressure; removing an aqueous liquid phase comprisingsolubilized hemicellulose from the pretreated feedstock, forming athickened pretreated hydrolyzate comprising the solid phase celluloseand lignin; introducing solid phase cellulose and lignin into acellulose hydrolysis zone wherein cellulose is contacted with acellulase and cellulose is enzymatically hydrolyzed to produce acellulose hydrolyzate slurry comprising an aqueous phase comprisingcellulose-derived fermentable sugars and a solid phase comprisinglignin; contacting the cellulose hydrolyzate slurry with a yeast toconvert cellulose-derived fermentable sugars to ethanol and form afermentation slurry comprising an aqueous phase comprising ethanol and asolid phase comprising lignin; distilling the fermentation slurry toproduce an ethanol rich product stream and a bottoms product comprisinga solid phase comprising lignin; and recovering a lignin-rich productfrom the bottoms product.

In another embodiment, the method comprises contacting the cellulosicbiomass feedstock with an acidic aqueous liquid medium to form anacid-impregnated cellulosic biomass feedstock; forming a pretreatedfeedstock comprising solubilized hemicellulose and a solid phasecomprising cellulose and lignin, the forming comprising contacting theacid-impregnated cellulosic biomass feedstock with H₂O at elevatedtemperature and pressure; removing an aqueous liquid phase comprisingsolubilized hemicellulose from the pretreated feedstock, forming athickened pretreated hydrolyzate comprising the solid phase celluloseand lignin; and introducing the thickened pretreated hydrolyzate into asaccharification and fermentation zone wherein solid phase cellulose anda cellulase are contacted to form cellulose-derived fermentable sugarsand at least a portion of the cellulose-derived fermentable sugars arecontacted with a yeast to convert cellulose-derived fermentable sugarsto ethanol, wherein the solid phase cellulose of the thickenedpretreated hydrolyzate is in the form of fibers such that at least about10% (by weight) of the fibers have a size in their largest dimensionless than about 1 mm.

In another embodiment, the method comprises contacting the cellulosicbiomass feedstock with an acidic aqueous liquid medium to form anacid-impregnated cellulosic biomass feedstock; forming a pretreatedfeedstock comprising solubilized hemicellulose and a solid phasecomprising cellulose and lignin, the forming comprising contacting theacid-impregnated cellulosic biomass feedstock with H₂O at elevatedtemperature and pressure; removing lignin from the pretreated feedstock;introducing solid phase cellulose into a cellulose hydrolysis zonewherein cellulose is contacted with a cellulase and cellulose isenzymatically hydrolyzed to produce a cellulose hydrolyzate slurrycomprising an aqueous phase comprising cellulose-derived fermentablesugars; contacting the cellulose hydrolyzate slurry with a yeast toconvert cellulose-derived fermentable sugars to ethanol and form afermentation slurry comprising an aqueous phase comprising ethanol; anddistilling the fermentation slurry to produce an ethanol rich productstream.

In a further embodiment, the method comprises contacting the cellulosicbiomass feedstock with an acidic aqueous liquid medium to form anacid-impregnated cellulosic biomass feedstock comprising cellulose,hemicellulose, and lignin; introducing a portion of the acid-impregnatedfeedstock into a microbe proliferation zone, and contacting cellulose,glucose, a nitrogen source, and a microbe that is effective to express acellulase enzyme in an aqueous biosynthesis medium within the microbeproliferation zone, thereby producing cellulase enzyme within theproliferation zone; introducing a portion of the acid-impregnatedfeedstock into a cellulose hydrolysis zone wherein cellulose iscontacted with a cellulase and cellulose is enzymatically hydrolyzed toproduce a cellulose hydrolyzate slurry comprising an aqueous phasecomprising cellulose-derived fermentable sugars; introducing at least aportion of the cellulase produced within the proliferation zone into thecellulose hydrolysis zone; contacting the cellulose hydrolyzate slurrywith a yeast to convert cellulose-derived fermentable sugars to ethanoland form a fermentation slurry comprising an aqueous phase comprisingethanol; and distilling the fermentation slurry to produce an ethanolrich product stream.

In a further embodiment, the method comprises pretreating the biomass toincrease the bioavailability of the hemicellulose and cellulosecontained therein; contacting the pretreated biomass with ahemicellulase to cause hemicellulose to be hydrolyzed to soluble C₅sugar(s) and produce a hemicellulase hydrolyzate slurry comprising anaqueous phase containing C₅ sugar(s) and a solid phase comprisingcellulose and lignin; separating an aqueous phase C₅ hydrolyzatefraction comprising C₅ sugar(s) from the hemicellulase hydrolyzateslurry, yielding a thickened residual fraction comprising a cake orconcentrated slurry comprising the solid phase cellulose and lignin;contacting C₅ sugars obtained in the aqueous phase C₅ fraction with ayeast, thereby converting C₅ sugar(s) to ethanol and producing a C₅fermentate containing ethanol; removing lignin from the thickenedresidual fraction; and contacting cellulose of the thickened fractionhaving lignin removed therefrom with a cellulase, thereby convertingcellulose to C₆ sugar(s) and producing a C₆ hydrolyzate.

In another embodiment, the method comprises contacting the cellulosicbiomass feedstock with an acidic aqueous liquid medium to form anacid-impregnated cellulosic biomass feedstock comprising cellulose,hemicellulose, and lignin; introducing a portion of the acid-impregnatedfeedstock into a microbe proliferation zone, and contacting cellulose,glucose, a nitrogen source, and a microbe that is effective to express acellulase enzyme in an aqueous biosynthesis medium within the microbeproliferation zone, thereby producing cellulase enzyme within theproliferation zone; removing lignin from the acid-impregnated cellulosicbiomass feedstock; introducing solid phase cellulose of theacid-impregnated cellulosic biomass feedstock having lignin removedtherefrom into a cellulose hydrolysis zone wherein cellulose iscontacted with a cellulase and cellulose is enzymatically hydrolyzed toproduce a cellulose hydrolyzate slurry comprising an aqueous phasecomprising cellulose-derived fermentable sugars; and introducing atleast a portion of the cellulase produced within the proliferation zoneinto the cellulose hydrolysis zone.

In another embodiment, the method comprises pretreating the biomass toincrease the bioavailability of the hemicellulose and cellulosecontained therein; contacting the pretreated biomass with ahemicellulase to cause hemicellulose to be hydrolyzed to soluble C₅sugar(s) and produce a hemicellulase hydrolyzate slurry comprising anaqueous phase containing C₅ sugar(s) and a solid phase comprisingcellulose and lignin; separating an aqueous phase C₅ hydrolyzatefraction comprising C₅ sugar(s) from the hemicellulase hydrolyzateslurry, yielding a thickened residual fraction comprising a cake orconcentrated slurry comprising the solid phase cellulose and lignin;contacting C₅ sugars obtained in the aqueous phase C₅ fraction with ayeast, thereby converting C₅ sugar(s) to ethanol and producing a C₅fermentate containing ethanol; introducing a portion of the thickenedresidual fraction into a microbe proliferation zone, and contactingcellulose, glucose, a nitrogen source, and a microbe that is effectiveto express a cellulase enzyme in an aqueous biosynthesis medium withinthe microbe proliferation zone, thereby producing cellulase enzymewithin the proliferation zone; and contacting cellulose of the thickenedfraction with a cellulase, thereby converting cellulose to C₆ sugar(s)and producing a C₆ hydrolyzate, wherein at least a portion of thecellulase is cellulase enzyme produced within the proliferation zone.

In a further embodiment, the method comprises pretreating the biomass toincrease the bioavailability of the hemicellulose and cellulosecontained therein; contacting the pretreated biomass with ahemicellulase to cause hemicellulose to be hydrolyzed to soluble C₅sugar(s) and produce a hemicellulase hydrolyzate slurry comprising anaqueous phase containing C₅ sugar(s) and a solid phase comprisingcellulose and lignin; separating an aqueous phase C₅ fraction comprisingC₅ sugar(s) from the hemicellulase hydrolyzate slurry, yielding athickened residual fraction comprising a cake or concentrated slurrycomprising the solid phase cellulose and lignin; contacting C₅ sugarsobtained in the aqueous phase C₅ fraction with a yeast, therebyconverting C₅ sugar(s) to ethanol and producing a C₅ fermentatecontaining ethanol; contacting cellulose of the thickened fraction witha cellulase, thereby converting cellulose to C₆ sugar(s) and producing aC₆ hydrolyzate slurry comprising C₆ sugars and a solid phase comprisinglignin; contacting the C₆ hydrolyzate slurry with a yeast to convert C₆sugars to ethanol and form a fermentation slurry comprising an aqueousphase comprising ethanol and a solid phase comprising lignin; distillingthe fermentation slurry to produce an ethanol rich product stream and abottoms product comprising a solid phase comprising lignin; andrecovering a lignin-rich product from the bottoms product.

In a further embodiment, the method comprises removing from the biomassfeedstock a fine particulate fraction, thereby forming a cleanedparticulate biomass feedstock; contacting the cleaned particulatebiomass feedstock with an acidic aqueous liquid medium to form anacid-impregnated cellulosic biomass feedstock; forming a pretreatedfeedstock comprising solubilized hemicellulose and a solid phasecomprising cellulose and lignin, said forming comprising contacting theacid-impregnated cellulosic biomass feedstock with H₂O at elevatedtemperature and pressure; removing lignin from the pretreated feedstock;introducing solid phase cellulose into a cellulose hydrolysis zonewherein cellulose is contacted with a cellulase and cellulose isenzymatically hydrolyzed to produce a cellulose hydrolyzate slurrycomprising an aqueous phase comprising cellulose-derived fermentablesugars; contacting the cellulose hydrolyzate slurry with a yeast toconvert cellulose-derived fermentable sugars to ethanol and form afermentation slurry comprising an aqueous phase comprising ethanol; anddistilling the fermentation slurry to produce an ethanol rich productstream.

In another embodiment, the method comprises removing from the biomassfeedstock a fine particulate fraction, thereby forming a cleanedparticulate biomass feedstock; contacting the cleaned particulatebiomass feedstock with an acidic aqueous liquid medium to form anacid-impregnated cellulosic biomass feedstock comprising cellulose;introducing a portion of the acid-impregnated feedstock into a microbeproliferation zone, and contacting cellulose, glucose, a nitrogensource, and a microbe that is effective to express a cellulase enzyme inan aqueous biosynthesis medium within the microbe proliferation zone,thereby producing cellulase enzyme within said proliferation zone;introducing a portion of the acid-impregnated feedstock into a cellulosehydrolysis zone wherein cellulose is contacted with a cellulase andcellulose is enzymatically hydrolyzed to produce a cellulose hydrolyzateslurry comprising an aqueous phase comprising cellulose-derivedfermentable sugars; introducing at least a portion of the cellulaseproduced within said proliferation zone into said cellulose hydrolysiszone; contacting the cellulose hydrolyzate slurry with a yeast toconvert cellulose-derived fermentable sugars to ethanol and form afermentation slurry comprising an aqueous phase comprising ethanol; anddistilling the fermentation slurry to produce an ethanol rich productstream.

In a still further embodiment, the method comprises removing from thebiomass feedstock a fine particulate fraction, thereby forming a cleanedparticulate biomass feedstock; pretreating the cleaned particulatebiomass feedstock to increase the bioavailability of the hemicelluloseand cellulose contained therein; contacting the pretreated biomass witha hemicellulase to cause hemicellulose to be hydrolyzed to soluble C₅sugar(s) and produce a hemicellulase hydrolyzate slurry comprising anaqueous phase containing C₅ sugar(s) and a solid phase comprisingcellulose and lignin; separating an aqueous phase C₅ hydrolyzatefraction comprising C₅ sugar(s) from the hemicellulase hydrolyzateslurry, yielding a thickened residual fraction comprising a cake orconcentrated slurry comprising said solid phase cellulose and lignin;contacting C₅ sugars obtained in said aqueous phase C₅ fraction with ayeast, thereby converting C₅ sugar(s) to ethanol and producing a C₅fermentate containing ethanol; removing lignin from the thickenedresidual fraction; and contacting cellulose of said thickened fractionhaving lignin removed therefrom with a cellulase, thereby convertingcellulose to C₆ sugar(s) and producing a C₆ hydrolyzate.

In another embodiment, the method comprises contacting the cellulosicbiomass feedstock with an acidic aqueous liquid medium to form anacid-impregnated cellulosic biomass feedstock comprising cellulose,hemicellulose, and lignin; introducing a portion of the acid-impregnatedfeedstock into a microbe proliferation zone, and contacting cellulose,glucose, a nitrogen source, and a microbe that is effective to express acellulase enzyme in an aqueous biosynthesis medium within the microbeproliferation zone, thereby producing cellulase enzyme within theproliferation zone; introducing solid phase cellulose into a cellulosehydrolysis zone wherein cellulose is contacted with a cellulase andcellulose is enzymatically hydrolyzed to produce a cellulose hydrolyzateslurry comprising an aqueous phase comprising cellulose-derivedfermentable sugars; introducing at least a portion of the cellulaseproduced within the proliferation zone into the cellulose hydrolysiszone; contacting the cellulose hydrolyzate slurry with a yeast toconvert cellulose-derived fermentable sugars to ethanol and form afermentation slurry comprising an aqueous phase comprising ethanol and asolid phase comprising lignin; distilling the fermentation slurry toproduce an ethanol rich product stream and a bottoms product comprisinga solid phase comprising lignin; and recovering a lignin-rich productfrom the bottoms product.

The present invention is further directed to a distiller's biomassproduct prepared from a cellulosic biomass feedstock comprisingcellulose, hemicellulose, lignin, and protein. In one embodiment, theweight ratio of the protein content of the biomass product to theprotein content of the biomass feedstock is at least 1:1.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a process flow of one embodiment of an ethanol productionprocess of the present invention.

FIG. 1A depicts a dry cleaning method of the present invention.

FIG. 2 depicts a pretreatment process of the present invention.

FIG. 3 depicts a process flow of another embodiment of an ethanolproduction process of the present invention.

Corresponding reference characters indicate corresponding partsthroughout the drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Described herein are improved processes for production of ethanol fromlignocellulosic biomass including, for example, processes which provideimproved ethanol yield. Also described herein are processes whichprovide various advantageous co-products. As detailed herein,improvements in ethanol yield and/or advantageous co-products may beprovided by one or more aspects of various protocols for treatment oflignocellulosic biomass that may be utilized in an ethanol productionprocess.

For example, various protocols for pretreatment of lignocellulosicbiomass have been observed to improve process efficiencies. Generally,these pretreatment protocols comprise contacting a lignocellulosicbiomass feedstock with an acidic liquid medium under certain conditions(e.g., certain mass ratios of acid to biomass feed). As used herein, theterm “pretreatment” refers to processing of biomass feedstock prior tohydrolysis of the lignocellulosic biomass for the primary purpose ofproducing fermentable sugars by hydrolysis of hemicellulose and/orcellulose.

Various embodiments of the present invention involve pretreatmentprotocols that utilize a feedstock and/or provide a pretreated feedstockhaving relatively high solids content (i.e., low moisture content).Various other protocols provide prescribed manners for contact of thefeedstock and the acidic liquid medium (e.g., by soaking or spraying)that promote advantageous dispersion of the acidic liquid mediumthroughout the biomass feedstock. These and other pretreatment protocolsmay also utilize methods for removal of one or more volatile componentsby venting during contact of acid-impregnated biomass feedstock andsteam at elevated temperature and pressure. By way of further example,one or more parameters of the pretreatment protocol are controlledand/or selected to provide a pretreated biomass feedstock having minimaltemperature variation. Further in accordance with the present invention,pretreated feedstock may be subjected to a conditioning operation toremove one or more components of the pretreated feedstock that mayinhibit fermentation of sugars derived from hemicellulose and/orcellulose.

In accordance with various embodiments of the present invention, thebiomass feedstock is subjected to a cleaning operation for purposes ofproviding a cleaned biomass feedstock suitable for effective acidimpregnation. In particular, various methods detailed herein areeffective for providing a cleaned biomass feedstock having a significantportion of impurities (e.g., components of the ash fraction of thebiomass feedstock) removed therefrom. As detailed elsewhere herein,these methods provide biomass feedstocks that provide advantageousconsumption of the acid by the feedstock as evidenced by, for example,advantageous fermentable sugar yields during pretreatment.Advantageously, these methods are conducted in the absence of wash waterand, thus, are referred to herein as “dry cleaning” methods.

The present invention is further directed to enzymatic hydrolysis ofhemicellulose-derived sugars prior to and/or in parallel with hydrolysisof fermentable cellulose-derived sugars, which has also been observed tocontribute to improved processes (e.g., improved ethanol yields). Forexample, various aspects of the present invention are directed tomethods for production of ethanol that include recovery ofhemicellulose-derived sugars and their conversion to ethanol along withrecovery of cellulose-derived sugars and their conversion to ethanol.

By way of further example, various aspects of the present invention aredirected to cellulase enzyme generation integrated into a process forpreparing ethanol from lignocellulosic biomass. For example, aspects ofthe present invention are directed to conversion of cellulose to glucoseby methods that include producing a cellulase enzyme within aproliferation zone and contacting the cellulase enzyme thus producedwith cellulose to generate cellulose-derived sugars (i.e., glucose). Invarious embodiments, integrated cellulase generation is combined alongwith recovery of hemicellulose-derived sugars and their conversion toethanol.

The present invention is also directed to protein-rich distiller'sbiomass products and lignin-rich co-products. More particularly,processes of the present invention are directed to recovery oflignin-rich co-products from biomass feedstock prior to production offermentable sugars by hydrolysis of cellulose-derived sugars. Processesof the present invention are likewise directed to recovery oflignin-rich co-products after production of ethanol fromcellulose-derived sugars. These processes for recovery of lignin-richco-products may be combined with recovery of hemicellulose-derivedsugars and their conversion to ethanol, either before or after recoveryof the lignin-rich co-product. Further in accordance with the presentinvention, recovery of lignin-rich co-products may be combined alongwith integrated cellulase generation.

I. Feedstock

Generally, the feedstock (1 in FIG. 1) comprises woody and/or non-woodycellulosic biomass provided by, for example, plant biomass, agriculturalwastes, forestry residues, and sugar processing residues. Moreparticularly, the feedstock may comprise grasses, such as switchgrass,cord grass, rye grass, reed canary grass, miscanthus, or combinationsthereof Additionally or alternatively, the feedstock may includeagricultural wastes such as rice straw, rice hulls, barley straw, corncobs, wheat straw, canola straw, oat straw, oat hulls, corn fiber,stover (e.g., sorghum, soybean stover and/or corn stover), orcombinations thereof. Suitable sugar-processing residues include, forexample, sugar cane bagasse, sweet sorghum, beet pulp, and combinationsthereof In various embodiments the feedstock comprises a non-woodybiomass selected from the group consisting of corn stover, wheat straw,barley straw, sorghum, switchgrass, miscanthus, and combinations thereofIn various preferred embodiments, the feedstock comprises corn stover.In these and other preferred embodiments, the feedstock comprises wheatstraw. Still further, in these and various other preferred embodiments,the feedstock comprises switchgrass. The feedstock may also include woodand forestry wastes such as, for example, recycled wood pulp fiber,sawdust, hardwood, softwood, forest thinnings, orchard thinnings, orcombinations thereof. Accordingly, in various embodiments, the feedstockcomprises a woody biomass.

Much of the following discussion, including the discussion belowregarding FIGS. 1 and 2, focuses on corn stover as the feedstock.However, unless specifically noted otherwise, it is to be understoodthat the following discussion generally applies to all suitablelignocellulosic biomass feedstocks.

Lignocellulosic biomass is a mixture of carbohydrate polymers from plantcell walls (i.e., cellulose and hemicellulose), lignin, and variousother components (e.g., ash and sand). For example, corn stovertypically has a cellulose content of from about 30 wt % to about 40 wt%, a hemicellulose content of from about 20 wt % to about 30 wt %, and alignin content of from about 15 wt % to about 25 wt %. Corn stovertypically contains a portion of ash (e.g., at least about 3 wt %, fromabout 3 wt % to about 10 wt %, or from about 4 wt % to about 8 wt %). Amajor portion of the ash in corn stover is silica. Therefore, forexample, the silica content of corn stover is generally at least about 1wt % or at least 5 wt %, typically from about 1 wt % to about 7 wt % orfrom about 1 wt % to 5 wt % (e.g., from about 3 wt % to 5 wt %).

Wheat straw typically has a cellulose content of from about 30 wt % toabout 45 wt %, a hemicellulose content of from about 20 wt % to about 30wt %, and a lignin content of from about 15 wt % to about 25 wt %. Wheatstraw typically contains a portion of ash (e.g., at least about 3 wt %,from about 3 wt % to about 10 wt %, or from about 4 wt % to about 8 wt%). A portion of the ash in wheat straw is silica. Therefore, forexample, the silica content of wheat straw is generally at least about 1wt % and typically from about 1 wt % to about 7 wt %.

By way of further example, switchgrass typically has a cellulose contentof from about 30 wt % to about 38 wt %, a hemicellulose content of fromabout 22 wt % to about 30 wt %, and a lignin content of from about 16 wt% to about 22 wt %. Switchgrass also typically contains a minor portionof ash (e.g., from about 3 wt % to about 8 wt %, or from about 4 wt % toabout 6 wt %).

Woody biomass, for example, typically has a cellulose content of fromabout 30 wt % to about 55 wt %, a hemicellulose content of from about 20wt % to about 35 wt %, and a lignin content of from about 15 wt % toabout 25 wt %. Woody biomass typically contains a very minor portion ofash (e.g., less than about 5 wt %, less than about 2 wt %, from about0.1 wt % to about 5 wt %, or from about 0.1 wt % to about 2 wt %).Similarly, the silica content in woody biomass is generally very low,approaching zero in some species. For example, the silica content ofwoody biomass is generally less than about 0.2 wt % and typically fromabout 0.01 wt % to about 0.2 wt %.

Various processes for production of ethanol from lignocellulosic biomassutilize sources of cellulose that have been subjected to one or moreoperations prior to treatment as detailed herein to break down thecellulose-hemicellulose-lignin complex (i.e., complex) and providefermentable sugars (e.g., acid impregnation followed by steamtreatment). For example, sugar cane bagasse is typically processed toprovide a slurry comprising biomass feedstock from which a substantialportion, if not substantially all the soluble components have beenremoved. These treatments may also solubilize hemicellulose, therebyreducing fermentable sugar yield during later processing. In addition,prior treatment of the bagasse typically provides a moisture-impregnatedsubstrate. A moisture-impregnated substrate impedes acid impregnation(detailed elsewhere herein).

Methods of the present invention are suitable for treatment of biomassfeedstock prior to any processing that will impact later processing forthe purposes of deriving fermentable sugars and/or increasing thebioavailability of cellulose. Thus, biomass feedstock treated by thepresent methods may be referred to as field-harvested or virginfeedstock. In contrast to feedstock subjected to prior treatment (e.g.,sugar cane bagasse) as described above, soluble components remain in thefeedstock. As detailed herein, solubilized hemicellulose providesfermentable sugars that contribute to ethanol yields. In this manner,maximum fermentable sugar yields and/or improvements in cellulosebioavailability may be provided by the present methods Inhibitors ofbreak down of the complex and/or enzymatic hydrolysis may be present inthe field-harvested or virgin feedstock. Various strategies detailedherein address these issues to substantially minimize, and preferablyavoid any impact on complex break down and/or fermentable sugar yields.

Typically, lignocellulosic biomass is provided for processing in itscondition as stored and the precise properties of the biomass feedstockare not narrowly critical. Moisture content of the feedstock may varydepending on a variety of factors including, for example, the durationof storage prior to processing. For example, corn stover typically has amoisture content of from about 5 wt % to about 20 wt % or from about 5wt % to about 15 wt %, preferably less than about 15 wt %, and even morepreferably less than 10 wt %. It is to be understood that moisturecontents provided herein refer to both free and bound moisture. If thefeedstock provided contains a relatively high moisture content (e.g.,greater than 20 wt %, or greater than about 25 wt %), the feedstock maybe heated prior to use to reduce its moisture content. However,feedstocks having moisture content within or below the above-notedranges are preferred. Heating of the feedstock prior to processingincreases the cost of the process. In addition, the energy requirementsof milling operations (detailed elsewhere herein) likewise increase asmoisture content of the feedstock increases.

Regardless of the moisture content, if the feedstock is stored atrelatively low temperatures it may be desired to heat the feedstockprior to treatment. For example, the rate of diffusion of acidthroughout the feedstock decreases with decreasing temperature, andheating relatively cold feedstock to temperatures that ensure sufficientdiffusion of acid during acid impregnation increases energy costs. Inparticular, during the winter months it is generally preferred topreheat frozen biomass feedstock to avoid rapid cooling of the diluteacidic liquid medium upon contact with the feedstock, which impedesdiffusion of acid throughout the feedstock (e.g., through formation ofan acid film on the surface of the feedstock). Also, relatively hot acidmay be used during acid impregnation, but generally does not overcomethe issues attendant relatively cold feedstock. Pre-heating thefeedstock may allow for a reduced temperature of the acidic liquidmedium. Thus, in various embodiments, the feedstock may be heated totemperatures up to about 40° C., up to about 50° C., or up to about 60°C. in an environment comprising an oxygen-containing gas (e.g., air orwaste gas such as boiler stack gas) to reduce the moisture contentand/or increase the temperature of the feedstock, e.g., to a temperaturein the range of from about 30° C. to about 60° C.

Generally, the feedstock (milled as described herein or as-provided forprocessing) contains particles of a size in their largest dimension ofless than about 6 cm (about 2.5 inches), less than about 5 cm (about 2inches), less than about 4 cm (about 1.5 inches), or less than about 2.5cm (about 1 inch). Typically, the feedstock contains particles of a sizefrom about 0.01 cm (about 0.004 inches) to about 6 cm (about 2.4inches), from about 0.1 cm (about 0.04 inches) to about 5 cm (about 2inches), or from about 0.5 cm (about 0.2 inches) to about 4 cm (about1.5 inches).

While not narrowly critical, the size of particulate feedstock mayimpact processing. For example, during acid-impregnation as detailedelsewhere herein, a significant portion of relatively large particlesmay provide relatively low exposed surface area for acid-biomasscontact. Accordingly, it is currently believed that a significantfraction of solids within the above-noted preferred ranges promotesimpregnation of the acid throughout the solids. If necessary to providesolids within the preferred range(s), the feedstock may be comminutedprior to processing to provide a feedstock of reduced and/or relativelyconsistent particle size (e.g., comprising particles within theabove-noted preferred ranges).

FIG. 1 depicts one embodiment of a process of the present invention and,in particular, describes a process for, inter alia, production ofethanol from corn stover (e.g., field-harvested, or virgin corn stover).The feedstock may be treated in a grinder, hammer mill or other suitablecomminuting device known in the art. As shown in FIG. 1, corn stover 1is introduced into milling apparatus 5 in which the feedstock is treatedto reduce the particle size of the feedstock material and produce amilled feedstock 9. If the feedstock is delivered in the form of bales,the bale wrap or strings are removed either manually or mechanicallyprior to grinding. The bale wrap or strings are commonly constructed ofpolypropylene or other plastic material which can interfere withprocessing of the biomass feedstock.

In accordance with various preferred embodiments, the particles of themilled feedstock may be described by various particle size parameters.For example, in various embodiments, milled feedstock comprisesparticles of a size distribution such that no more than about 40 wt %,no more than about 30 wt %, or no more than about 20 wt % of thefeedstock particles are retained by a #10 Sieve. Additionally oralternatively, milled feedstock suitable for use in the processes of thepresent invention may comprise particles of a size distribution such atleast about 60 wt %, at least about 70 wt %, or at least about 80 wt %of the feedstock particles are retained by a #60 Sieve.

A significant portion, or fraction of relatively fine feedstockparticles may be undesired due to their impact on processing of thefeedstock. For example, relatively fine particles may be lost duringfiltration and washing of feedstock particles, representing a loss incellulose and/or hemicellulose. Accordingly, in various preferredembodiments, a fraction of relatively fine feedstock particles isremoved prior to processing (i.e., prior to acid impregnation). Forexample, a fraction of feedstock particles comprising particles having asize in their largest dimension of less than about 100 microns may beremoved from the feedstock prior to processing. Fractions of relativelyfine particles may include a relatively high ash content (e.g., up to 40wt %). Thus, removal of such a portion of the feedstock likewise reducesthe ash proportion of the feedstock. As detailed elsewhere herein,removal of a fine particulate fraction provides a cleaned biomassfeedstock exhibiting one or more advantageous properties based on thereduced ash content. For example, in various preferred embodimentscleaned biomass feedstocks provide advantageous fermentable sugar yieldsduring pretreatment and/or enzymatic hydrolysis.

Milling, or grinding of the feedstock generally proceeds in accordancewith conventional methods known in the art. In various embodiments themilling or grinding operation is conducted as a single-step operation.In such an embodiment, feedstock (e.g., de-stringed bales of biomassfeedstock) is introduced into a milling apparatus suitable for providinga milled feedstock of the desired properties. For example, in variouspreferred embodiments suitable milling operations comprise grinding thefeedstock for passage through a screen having openings of a size rangingfrom about 1.25 cm (about 0.5 inches) to about 4 cm (about 1.5 inches)(e.g., about 2.5 cm (about 1 inch)).

One-step milling of feedstock has generally been observed to providefeedstock of suitable particle size distributions. However, such methodsmay suffer one or more disadvantages. For example, wear on the screensmay be accelerated due to the lack of removal of contaminants (e.g.,rocks, metal, and other contaminants present in the virgin feedstock)prior to passing the feedstock over the screens. While the particle sizedistributions provided by one-step milling are generally suitable, thedistributions may be wider than those provided by other methods. Invarious other preferred embodiments, milling of the feedstock proceedsvia a two-step process. In such processes, feedstock (e.g., de-stringedbales of feedstock) is first subjected to relatively coarse sizereduction and passed over screens for the primary purpose of removinglarger particles (including contaminants) from the feedstock. Screenssuitable for the first step of a two-step milling operation typicallycomprise openings of a size of from about 8 cm (about 3 inches) to about12.5 cm (about 5 inches). The second step utilizes smaller screens forthe purpose of isolating and recovering feedstock particles within thedesired particle size distribution including, for example, screensdescribed above in connection with one-step milling. It is currentlybelieved that two-step milling comprising removal of larger particlesprior to final milling provides for recovery of milled feedstock that isnot only within the desired particle size distribution but has anarrower particle size distribution than is obtained in the single stepprocess. However, this advantage may be offset by the increased costassociated with the additional processing. One skilled in the art mayselect an appropriate milling protocol based on, for example, theproperties of the virgin, or untreated feedstock and the desiredproperties of the milled feedstock.

Regardless of whether a one-step or two-step milling operation isutilized, contaminants (e.g., rocks and/or metal) may be removed fromthe feedstock by passing the feedstock over magnets during, between, orafter milling operations. For example, in those embodiments in which atwo-step milling operation is utilized, feedstock is passed over magnetsbetween milling operations.

In various embodiments, the feedstock (e.g., milled corn stover) may besubjected to a cleaning operation prior to further treatment to removevarious impurities and contaminants (e.g., rock, dirt, sand, and othertramp materials) and feedstock particles of undesired size. Cleaning ofthe milled feedstock proceeds generally as known in the art including,for example, by a process comprising passing the feedstock over asuitable screen that separates desired and undesired particles.Typically, desired and undesired particles are separated by vibrationand/or shaking of the screen. Contaminants (e.g., ferrous contaminants)and oversized particles and fines may also be removed by magneticseparation. Contaminants may also be removed from the feedstock bycontact with a suitable flow of air (i.e., air classification) and/orcontact with an aqueous washing medium (e.g., water). Water washing ofthe feedstock has been observed to be effective for removal of variousimpurities (e.g., soil and sand). However, water washing may beundesired since it may provide a relatively moist pretreated feedstockfor acid impregnation, which may be undesired because it may hinderdispersion of the acid throughout the feedstock. Again with reference toFIG. 1, milled feedstock 9 is introduced into vessel 13 to form a wastestream 17 and milled and cleaned feedstock 21. In various preferredembodiments, contaminants are removed using a combination of one or moreof the above-noted methods (e.g., air classification and magneticseparation).

In accordance with the process depicted in FIG. 1, milled and cleanedcorn stover 21 generally has a total solids content of at least about 70wt %, or at least about 80 wt % (e.g., about 90 wt %). The milled andcleaned corn stover is generally stored and/or processed underrelatively mild to warm ambient conditions (e.g., a temperature ofapproximately 20° C. and atmospheric pressure).

Generally in accordance with the present invention, and with referenceto the process depicted in FIG. 1, the solid portion of the cleaned andmilled feedstock typically comprises a significant fraction of sugarsincluding, for example, various polysaccharides such as glucan, xylan,arabinan, mannan, and galactan, various monosaccharides such as xyloseand glucose, and combinations thereof. For example, in variousembodiments, the total glucan content of the milled and cleaned cornstover is typically from about 30 wt % to about 45 wt % (dry weightbasis), more typically from about 35 wt % to about 42 wt % and, stillmore typically, from about 37 wt % to about 40 wt %. In these andvarious other embodiments, the total xylan content of the milled andcleaned corn stover is typically from about 10 to about 25 wt % (dryweight basis), more typically from about 15 to about 25 wt % and, stillmore typically, from about 18 to about 22 wt %. Additionally oralternatively, the arabinan content of the milled and cleaned cornstover is typically from about 1 to about 5 wt % (dry weight basis),more typically from about 2 to about 4.0 wt % and, still more typically,from about 2.5 to about 3.5 wt %.

The lignin content of the milled and cleaned corn stover is typicallyfrom about 10 to about 25 wt % (dry weight basis), more typically fromabout 15 to about 25 wt % and, still more typically, from about 18 toabout 23 wt %.

An ash portion of the milled and cleaned corn stover typicallyconstitutes from about 2 to about 8 wt % (dry weight basis), moretypically from about 3 to about 6 wt % and, still more typically, fromabout 4 to about 5 wt % of the milled and cleaned corn stover. Thecleaned and milled corn stover also typically comprises minorproportions (e.g., from about 5 to about 12 wt %, or about 8 wt %) ofvarious other components (e.g., acetate, uronic acid, and protein).

II. Pretreatment

Various methods for deriving fermentable sugars (e.g., glucose) fromlignocellulosic biomass include acid hydrolysis utilizing relativelyconcentrated acids (e.g., acids having an acid content of up to 70 wt %,or greater) to dissolve and hydrolyze to glucose the cellulose componentof the biomass. These methods typically provide suitable glucose yields,but generally suffer from one or more disadvantages. For example,concentrated acids require the use of specialized equipment and precisecontrol of moisture in the system. In addition, sugars produced via theacid-catalyzed hydrolysis are often degraded by the relatively harshhydrolysis conditions. For example, cellulose may ultimately behydrolyzed to produce hydroxymethylfurfural rather than glucose, whichmay be further degraded to produce levulinic acid or formic acid. Inaddition, xylose produced by hydrolysis of hemicellulose may be degradedto produce furfural, tars and various other degradation products (e.g.,condensation compounds associated with and/or derived from lignin).

To avoid one or more of these disadvantages, pretreatment methods havebeen developed that utilize a relatively dilute acid (e.g., acidicliquid media containing less than 5 wt % acid). Rather than hydrolysisof cellulose and/or hemicellulose to produce fermentable sugars, theprimary purpose of dilute acid treatment (often referred to herein asacid impregnation, or pretreatment) is preparation of the feedstock forsubsequent enzymatic hydrolysis to produce fermentable sugars. Forexample, as detailed elsewhere herein, pretreatment protocols combiningdilute acid treatment and treatment of the acid-impregnated feedstock atelevated temperature and pressure (referred to elsewhere herein as steamtreatment, or steam explosion) degrade, or break down thecellulose-hemicellulose-lignin complex of the biomass. In this manner,the cellulose is more susceptible to enzymatic hydrolysis to producefermentable sugars. Increasing the susceptibility of cellulose toenzymatic hydrolysis is generally referred to herein as increasing thebioavailability or digestibility of the cellulose. Such pretreatmentprotocols also typically result in solubilizing at least a portion(e.g., up to or in excess of 50%) of the hemicellulose. Solubilizinghemicellulose increases the availability of cellulose to cellulaseenzymes and provides hemicellulose that may be hydrolyzed to producefermentable sugars. A further advantage of increased cellulosebioavailability is a reduction in the proportion of cellulase enzymerequired to provide suitable yields of cellulose-derived fermentablesugars.

A. Acid Impregnation

Again with reference to FIG. 1, milled and cleaned feedstock (cornstover) 21 is introduced into acid impregnation vessel 25. Acid 29introduced into acid impregnation vessel 25 typically comprises an acidselected from the group consisting of hydrochloric acid, sulfuric acid,sulfurous acid, sulfur dioxide, nitric acid, and combinations thereof.As noted, the primary purpose of acid impregnation is preparation of thefeedstock for enzymatic hydrolysis to produce fermentable sugars. Thatis, the primary purpose of acid impregnation is increasing thebioavailability of the feedstock, rather than hydrolysis of celluloseand/or hemicellulose to produce fermentable sugars. Accordingly, acid 29is typically in the form of a relatively dilute acid. More particularly,in accordance with the process depicted in FIG. 1, acid 29 is typicallyin the form of an acidic liquid medium having an acid concentration ofless than about 5 wt %, less than about 4 wt %, or less than about 3 wt%. For example, typically the clean milled corn stover is contacted withan acidic liquid medium including an acid at a concentration of fromabout 0.2 wt % to about 4.5 wt %, preferably from about 0.7 wt % toabout 3.5 wt % and, more preferably, from about 1.0 wt % to about 3.0 wt% (e.g., from about 2.0 wt % to about 2.5 wt %). Regardless of theprecise composition of the acidic liquid medium, typically the biomassfeedstock is contacted with (i.e., the uptake of acid by the feedstock)at least about 0.005 kg acid (e.g., H₂SO₄ or HCl) (acid weight basis)per kg feedstock (dry weight basis), or at least about 0.01 kg acid perkg feedstock. Preferably, the acid uptake by the feedstock is from about0.01 kg to about 0.05 kg acid per kg of feedstock, more preferably fromabout 0.02 kg to about 0.04 kg acid per kg of feedstock and, still morepreferably, from about 0.02 kg to about 0.03 kg acid per kg offeedstock. The solids content of acid-impregnated biomass generallyranges from 25 wt % and 50 wt %, or from about 30 wt % to about 45 wt %.

The precise configuration of acid impregnation vessel 25 is not narrowlycritical and may be readily selected from suitable apparatus known inthe art. For example, acid impregnation as detailed herein may beconducted in a batch reactor (e.g., a stirred-tank reactor), or a batchmixer (e.g., pug mixer, paddle mixer, ribbon mixer), or may be conductedin a vessel suitable for continuous operation (e.g., a continuousstirred-tank reactor or plug flow reactor), or a continuous mixer (e.g.,pug mixer, paddle mixer, ribbon mixer, mixing screw).

The temperature of the acid 29 introduced into acid impregnation vessel25 and/or the mixture of biomass feedstock and acid is generally atleast about 30° C., at least about 40° C., or at least about 50° C. Forexample, in various embodiments, the temperature of the acid is fromabout 20° C. about 95° C., or from about 30° C. to about 75° C.

The contact time for contact of the biomass feedstock and acid byspraying or soaking is typically from about 1 minute to about 15minutes, more typically from about 2 minutes to about 10 minutes and,more typically, from about 3 minutes to about 6 minutes. For contact byspraying, prior to further processing, the acid-sprayed corn stover istypically held in an insulated or heat-jacketed bin for from about 5minutes to about 60 minutes, from about 10 minutes to about 45 minutes,or from about 15 minutes to about 30 minutes. In the case of feedstockcontacted with the acid by soaking, the contact time is typicallyfollowed by a draining and dewatering step to remove excess acidsolution and to provide an acid-impregnated feedstock of suitable solidscontent (e.g., from about 30 to about 65 wt %) for introduction into thepretreatment reactor.

The total flow of milled and cleaned corn stover that may be treated bythe processes of the present invention and, in particular, the processdepicted in FIG. 1 is not narrowly critical. The total flow of milledand cleaned corn stover depends on a variety of factors including, forexample, the bulk density of the feedstock and the desired fill factorof the reactor. Generally, the total flow of milled and cleaned cornstover 21 introduced into the acid impregnation vessel (e.g., acontinuous acid spray impregnation vessel) is from about 20 to about 90pounds per hour-ft³ reactor volume (lb/hr-ft³ reactor volume), fromabout 30 to about 70 lb/hr-ft³ reactor volume, or from about 40 to about60 lb/hr-ft³ reactor volume. Depending on various factors including, forexample, the composition of the acidic liquid medium and/or the cleanedand milled feedstock, the total proportion of acidic liquid mediumintroduced into the acid impregnation vessel is generally from about 30to about 60 pounds per hour-ft³ reactor volume (lb/hr-ft³ reactorvolume), or from about 40 to about 50 lb/hr-ft³ reactor volume. It is tobe understood that the rates of introduction of feedstock and/or acidicliquid medium utilized in the process of the present invention are notnarrowly critical. These flows are provided to generally indicatesuitable flows, but it is currently believed that the processes of thepresent invention are likewise suitable for processes utilizingproportions of feedstock outside the specified ranges.

In the case of contact of the feedstock and acid by soaking, the totalsolids content in the acid impregnation vessel generally depends onvarious factors including, for example, the composition of the acidicliquid medium, the composition of the cleaned and milled feedstock,and/or the particle size distribution of the feedstock, but is generallyfrom about to about 4 wt % to about 12 wt %, from about 5 wt % to about10 wt %, or from about 5 wt % to about 7 wt %.

Contacting the biomass feedstock and an acidic liquid medium provides anacid-impregnated feedstock 33 in the form of a slurry comprising biomasssolids dispersed throughout the acidic liquid medium. Generally, thetemperature of the acid-impregnated corn stover reaches approximatelythe temperature of the acidic liquid medium contacted with the cornstover. That is, the temperature of the acid-impregnated corn stover istypically from about 20° C. about 95° C., or from about 40° C. to about80° C. (e.g., about 60° C.). Additionally, the pH of theacid-impregnated corn stover is preferably less than about 4, less thanabout 3, or less than about 2 (e.g., about 1).

As noted, rather than preparation of fermentable sugars, the primarypurpose of acid impregnation is increasing the bioavailability, orpretreatability of the feedstock. Accordingly, the composition of theacid-impregnated feedstock generally corresponds to the composition ofthe milled and cleaned feedstock, adjusted based on the presence of theacidic liquid medium dispersed throughout the feedstock. For example,the acid-impregnated feedstock 33 generally has a total solids contentof at least about 25 wt %, or at least about 30 wt % (e.g., at leastabout 35 wt %, at least about 40 wt %, or at least about 45 wt %).Typically, the solids content of the acid-impregnated feedstock is fromabout 30 to about 70 wt %, more typically from about 35 to about 55 wtand, still more typically, from about 40 to about 50 wt %.

Typically, the total glucan content of the acid-impregnated feedstock(e.g., corn stover) is from about 25 to about 50 wt % (dry weightbasis), more typically from about 30 to about 45 wt % and, still moretypically, from about 35 to about 40 wt %. In these and various otherembodiments, the total xylan content is typically from about 10 to about35 wt % (dry weight basis), more typically from about 15 to about 30 wt% and, still more typically from about 20 to about 25 wt %. Additionallyor alternatively, the arabinan content of the acid-impregnated feedstockis typically from about 1 to about 5 wt % (dry weight basis), moretypically from about 1.5 to about 4 wt % and, still more typically, fromabout 2 to about 3.5 wt %.

The lignin content of the acid-impregnated feedstock (e.g., corn stover)is typically from about 10 to about 25 wt % (dry weight basis), moretypically from about 10 to about 25 wt % and, still more typically, fromabout 15 to about 22 wt %.

The ash portion of the acid-impregnated feedstock 33 typicallyconstitutes from about 1 to about 8 wt % (dry weight basis), moretypically from about 2 to about 8 wt % and, still more typically, fromabout 3 to about 6 wt % of the milled and cleaned feedstock (e.g., cornstover). The acid-impregnated feedstock also typically comprises minorproportions of various other components (e.g., from about 1 to about 6wt % or from about 1 to about 4 wt % protein, from about 1 to about 4 wt% acetyl compounds, and from about 1 to about 4 wt % uronic acids).

As detailed below, the biomass solids may be contacted by soaking in theacidic liquid medium or by spraying liquid medium onto the feedstock.Each manner of contact of the dilute acid and feedstock providessuitable impregnation. But depending on the manner of contact utilized,various strategies may be employed to promote dispersion of the acidthroughout the feedstock. For example, as detailed below, when the acidis sprayed onto the feedstock, agitation or mixing may be employed topromote dispersion of the acid throughout the feedstock.

1. Soaking

For soaking of biomass feedstock, an appropriate proportion of an acidicliquid medium having an acid concentration and/or providing an acid tobiomass solids ratio noted above is typically selected. The amount ofliquid medium utilized may be readily selected by one skilled in the artdepending on the acid concentration, amount of feedstock to be treated,etc. For example, typically the feedstock and acid are contacted bysoaking the feedstock in at least about 10 kg acidic liquid medium perkg feedstock, or at least about 15 kg acidic liquid medium per kgfeedstock. Utilizing a relatively high proportion of acidic liquidmedium may allow utilizing dilute acids including the acids atconcentrations at or near the above-noted lower limits of acidconcentration (e.g., about 1.0 wt %) in view of the relatively highproportion of liquid medium contacted with the feedstock.

Soaking of the feedstock generally occurs for a time that promotessufficient dispersion of the acid throughout the biomass feedstocksolids. The duration of soaking may generally be selected based on theproperties of the feedstock, the desired acid content of the resultingslurry and/or moisture content of the resulting slurry. For example, thehold time of contact by soaking (i.e., time of contact between thefeedstock and acid prior to any further processing) is typically atleast about 1 minute, at least about 5 minutes, or at least about 10minutes (e.g., at least about 15 minutes, at least about 20 minutes, orat least about 25 minutes). While a suitable hold time is desired topromote impregnation of the feedstock, degradation of the fibers maybegin to occur as the hold time and temperature reach certain limits. Asused herein, degradation of feedstock fibers generally refers todissolving or hydrolysis of hemicellulose, rather than break-down of thecellulose-hemicellulose-lignin complex. Thus, excessive fiberdegradation reduces fermentable sugar yields and/or improvements incellulose bioavailability and, accordingly, is preferably minimized. Invarious preferred embodiments the hold time for soaking contact istypically from about 1 minute to about 60 minutes, more typically fromabout 3 minutes to about 30 minutes and, still more typically, fromabout 4 minutes to about 20 minutes.

Soaking of the biomass feedstock may provide a slurry of the feedstocksolids in the liquid medium having a relatively high moisture content.As detailed elsewhere herein, in various preferred embodiments, themoisture content of the acid-impregnated feedstock is below certainlevels, or within various preferred ranges. Typically, the soakedbiomass feedstock is dewatered to reduce its moisture content, ifnecessary. When contacting the biomass feedstock and acid by soaking,preferably the entire mass of feedstock is submerged in the acidicliquid medium. Submersion of the feedstock promotes bulk movement of thefeedstock and/or liquid medium to provide dynamic and continuous contactof the feedstock and acidic liquid medium. To promote dynamic physicalcontact of the feedstock and acidic liquid medium, the feedstock/acidicliquid medium slurry is typically agitated and preferably agitatedcontinuously. Agitation may be conducted using conventional apparatusknown in the art including, for example, agitators, mixers, and mixingconveyors, depending on the acid impregnation vessel being utilized.

2. Spraying

As noted, the feedstock and acid may also be contacted by spraying anacidic liquid medium onto the biomass feedstock. The precise manner ofspraying is not narrowly critical and is generally conducted inaccordance with means known in the art. As compared to soaking of thefeedstock in an acidic liquid medium, reduced proportions of acidicliquid medium are typically used for contact of the feedstock and acidicliquid by spraying. In this manner, material costs are reduced. Tocompensate for the reduction of proportion of liquid medium, approachesmay be taken to promote dispersion of the acid throughout the feedstock.

For example, generally the biomass feedstock is agitated while theacidic liquid medium is sprayed onto the feedstock and/or uponcompletion of spraying of the acidic liquid medium onto the feedstock.More particularly, the feedstock is agitated to distribute the acidicliquid medium throughout the feedstock and bring particles into contactwith other particles. In various preferred embodiments, agitationprovides mutually abrading contact of the particles and the resultingrubbing action between the particles promotes distribution of the acidicliquid medium throughout the feedstock. Typically, the feedstock isagitated for a period of from about 1 to about 10 minutes and, moretypically, for from about 2 to about 5 minutes. In certain preferredembodiments, the feedstock and acidic liquid medium are contacted in asuitable vessel comprising counter-rotating shafts that provideagitation of the feedstock in a manner that promotes distribution of themedium throughout the feedstock.

As detailed elsewhere herein, acid-impregnated feedstock preferablyexhibits moisture contents within various preferred ranges (e.g., lessthan about 70 wt % or less than about 50 wt %). Soaking of the biomasstypically provides a slurry having a moisture content that necessitatesdewatering to achieve such often-preferred moisture contents. Incontrast, spraying of dilute acid is controlled to prepareacid-impregnated feedstock that typically exhibits a moisture content atthe conclusion of the spraying step that falls within these preferredranges, thereby avoiding the need for dewatering. Accordingly, contactby spraying may be preferred in various embodiments.

As with contact by soaking, the biomass feedstock and acidic liquidmedium are generally contacted for a time sufficient to suitablydisperse the acid throughout the feedstock. Often, the reducedproportion of liquid medium utilized in contact by spraying may lead toincreased contact, or hold times for acid impregnation. For example, thehold time may be at least about 10 minutes, at least about 20 minutes,or at least about 40 minutes. However, preferably the reduced proportionof the acidic liquid medium is compensated for by agitation of thebiomass. Accordingly, typically the hold time for spraying contact is nomore than about 60 minutes, more typically no more than about 40 minutesand, still more typically, no more than about 20 minutes. In variouspreferred embodiments, the hold time is from about 2 to about 35minutes, from about 5 to about 30 minutes, or from about 10 to about 20minutes.

3. Wetting Agent

Regardless of its precise composition (e.g., moisture content) or itsmanner of contact with the feedstock (e.g., by soaking or spraying), theacidic liquid medium contacted with the feedstock may include asurfactant, or wetting agent to promote dispersion of the acidthroughout the resulting acid-impregnated biomass slurry. Moreparticularly, including a surfactant(s) in the acidic liquid medium mayreduce the surface tension of the liquid medium to promote dispersion ofthe liquid medium and acid contained therein throughout the biomassfeedstock. Suitable surfactants are generally bio-degradable andnon-toxic and generally include commercially available surfactants(e.g., various anionic, cationic, and nonionic surfactants). Based onthe lower proportion of acidic liquid medium utilized, use of a wettingagent during acid impregnation is often preferred in those embodimentsin which the feedstock is contacted with the acidic liquid medium byspraying.

Suitable anionic surfactants include alkyl sulfate salts, arylalkylsulphonates, fatty acid salts, and combinations thereof. For example,commercially available anionic surfactants include, for example, DOWFAXand TRITON (Dow Chemicals), BIO-TERGE (Stepan), and OT-A (Cytec).Suitable cationic surfactants include alkyl quaternary ammonium saltsincluding, for example, the commercially available PRAEPAGEN surfactants(Clariant). Suitable nonionic surfactants include alcohol ethoxylates(e.g., alkyl polyethylene oxides), alcohol propoxylates, alcoholethoxyalte-propoxylates, fatty alcohols, and combinations thereof.Commercially available nonionic surfactants include, for example,TERGITOL 15-S-12 and TRITON DF-16 (Dow Chemicals), SILWET L-77 (HelenaChemical Co.), and Activator 90 (Loveland Products, Inc.). In variousembodiments, nonionic surfactants are preferred as their performance isgenerally unaffected by the presence of an acidic liquid medium.

In addition to the above-noted surfactants, suitable wetting includevarious alcohols such as, for example, methanol, ethanol, propanol, andbutanol. Advantageously, alcohols suitable for use as surfactants may begenerated elsewhere in the process and recycled to the acid impregnationvessel.

Additionally or alternatively, in various preferred embodiments thebiomass feedstock and surfactant may be contacted prior to contact ofthe feedstock and acid. For example, feedstock (optionally subjected tomilling and/or a cleaning operation) may be contacted with an amount ofwetting agent prior to acid impregnation. The feedstock may be contactedwith the wetting agent by soaking the feedstock in a suitable proportionof wetting agent or liquid medium comprising a suitable proportion ofwetting agent. Preferably, the feedstock and wetting agent are contactedby spraying onto the feedstock a suitable portion of wetting agent or aliquid medium comprising the wetting agent. Although contact of thefeedstock and wetting agent disperses wetting agent throughout thefeedstock, dispersion of the wetting agent throughout the feedstockoccurs primarily during dispersion of the acid throughout the feedstockduring acid impregnation.

4. Heating During Acid Impregnation

In various embodiments, heating during contact of the biomass feedstockand dilute acid is employed to promote dispersion of the acid throughoutthe resulting acid impregnated biomass slurry. Typically, any heatingfor this purpose involves heating the biomass feedstock/dilute acidmixture to temperatures of at least about 10° C., at least about 20° C.,or at least about 40° C. However, solubilization of the hemicellulosecomponent of the biomass feedstock preferably does not occur to anysignificant degree during acid impregnation but, rather, preferablyoccurs during subsequent processing (e.g., steam pretreatment and/orenzymatic hydrolysis as detailed elsewhere herein). Accordingly, thetemperature during acid impregnation and any heating of the feedstockand dilute acid associated therewith is preferably controlled tominimize, and preferably avoid solubilization of hemicellulose. Based onthe foregoing, temperatures of acid impregnation (and any associatedheating) are preferably maintained at no more than about 100° C., nomore than about 90° C., or no more than about 80° C. Thus, in accordancewith the foregoing, preferably the temperature during acid impregnationis from about 10° C. to about 100° C., more preferably from about 30° C.to about 90° C. and, still more preferably, from about 40° C. to about80° C.

In addition to contributing to solubilization of hemicellulose,contacting the feedstock with moisture prior to contact with acid may beundesired since the moisture may be dispersed throughout the feedstockand inhibit dispersion of acid throughout the biomass feedstock and/orresult in relatively uneven acid dispersion throughout the biomassfeedstock. Thus, in accordance with those embodiments in which thefeedstock is heated during acid impregnation, the feedstock is heated inthe presence of a relatively low moisture environment. Generally, thefeedstock is heated in the presence of an environment having a relativehumidity of less than about 100%, or less than about 80%. In accordancewith various preferred embodiments, the feedstock is heated in thepresence of air heated to a temperature of at least about 20° C., or atleast about 40° C. The feedstock may also be heated by contact with aflue gas at such temperatures.

5. Steaming Prior to Acid Impregnation

Although desired in certain situations including, for example, when theincoming biomass feedstock is stored at relatively low temperatures suchas during the winter months, heating of the biomass feedstock is oftenundesired as it increases energy and operating costs. Thus, rather thanheating, in various embodiments the biomass feedstock is contacted withsteam prior to contact with acid. That is, in various preferredembodiments is subjected to a pre-steaming operation prior to acidimpregnation. Pre-steaming of the biomass feedstock provides variousadvantages. For example, pre-steaming removes regions throughout thebiomass feedstock largely made up of air pockets that during acidimpregnation will provide uptake of the acid, but do not contribute touptake of acid by feedstock particles. Injecting steam throughout thefeedstock removes aerified portions of the feedstock and thesteam-containing regions of the feedstock are removed when thesteam-infused feedstock is contacted with the acidic liquid medium.Removal of these steam pockets upon contact of the feedstock structurewith the acidic liquid medium causes the feedstock to collapse andprovide a feedstock with a substantial portion, and preferably near allof the air pockets that do not contribute to effective acid impregnationremoved. In this manner, pre-steaming provides a biomass feedstock thatpromotes more effective acid impregnation by removal of regionsthroughout the biomass feedstock mass that do not contribute to uptakeof the acid by feedstock particles. Pre-steaming also allows for use ofan acidic liquid medium at lower temperatures as compared to liquidmedia typically used in the connection with feedstocks that have notbeen subjected to pre-steaming Pre-steaming may be conducted inaccordance with methods and utilizing apparatus generally known in theart including, for example, as described in U.S. Pat. Nos. 3,383,277 and4,746,404, the entire contents of which are incorporated by referencefor all relevant purposes.

Generally during pre-steaming, the biomass feedstock is contacted withsteam in a suitable vessel or reactor. Typically, the biomass feedstockis contacted with steam introduced into the vessel under a steampressure of about 5 psig and steam temperature of about 110° C., a steampressure of about 10 psig and a steam temperature of about 115° C., or asteam pressure of about 15 psig and steam temperature of about 120° C.Typically, the biomass feedstock is contacted with steam at atemperature of from about 100 to about 130° C., from about 100 to about120° C., or from about 100 to about 110° C. Generally, steam isintroduced into the vessel at a rate of at least about 10 kg/hour, orfrom about 10 to about 20 kg/hour. Although not narrowly critical,generally pre-steaming is conducted for a time of from about 5 to about30 minutes, or from about 10 to about 20 minutes. In variousembodiments, a minor portion of the dilute acid may be introduced intothe pre-steaming vessel. For example, an acidic liquid medium containingan acid at a concentration of less than about 3 wt %, less than about 2wt %, less than about 1 wt %, or less than about 0.5 wt % may beintroduced into the pre-steaming vessel. The total proportion of acidicliquid medium is not narrowly critical and generally depends on themanner of contact of the feedstock and acidic liquid medium. Forexample, the acidic liquid medium and feedstock may be contacted at aproportion of from about 0.5 to about 15 g acidic liquid medium per gramfeedstock introduced into the pre-steaming vessel.

6. Ash Removal

While acid-impregnation in accordance with the foregoing description hasbeen found to suitably impregnate the feedstock, various treatmentsprior to impregnation may be employed to improve the efficiency of thisoperation. For example, ash present in the feedstock may consume theacid (e.g., by reaction to form salts) and, thus, reduce impregnation ofthe acid throughout the feedstock. As the proportion of ash approachesand/or exceeds the above-noted upper limits of ash concentration, ashremoval may be preferred prior to acid impregnation. Removal of ash fromthe feedstock may be conducted by, for example, washing of theacid-impregnated feedstock as detailed elsewhere herein, or by passingthe feedstock over a suitable screen for removal of fines and looseparticles (e.g., dirt).

Generally, the ash portion of the biomass feedstock includes variouscomponents such as silica, calcium-containing components,magnesium-containing components, sodium-containing components,potassium-containing components, phosphorus-containing components,aluminum-containing components, and combinations thereof Typically, theash content of the biomass feedstock is from about 1 wt % to about 10 wt% or from about 3 wt % to about 10 wt % for non-woody biomass, or fromabout 0.1 wt % to about 5 wt % or from about 0.1 wt % to about 2 wt %for woody biomass. The ash content of biomass feedstock generallyincludes an acid soluble fraction and an acid insoluble fraction. Theacid insoluble ash fraction generally comprises silica. Typically,silica constitutes the major portion of the insoluble fraction andtypically constitutes at least about 1 wt %, or at least about 3 wt % ofthe biomass feedstock. Generally, the acid soluble ash fractionconstitutes at least about 30 wt %, or at least about 40 wt % (e.g.,from about 40 wt % to about 50 wt % of the ash of the feedstock). Theacid insoluble ash fraction typically constitutes at least about 50 wt%, or at least about 60 wt % (e.g., from about 50 wt % to about 60 wt %of the ash of the feedstock).

For example, corn stover typically has an ash content of at least about3 wt % or from about 3 wt % to about 10 wt %. Additionally oralternatively, corn stover typically has a silica content of at leastabout 1 wt %, at least about 3 wt %, or at least about 5 wt % (e.g.,from about 1 wt % to about 7 wt % or from about 1 wt % to 5 wt %, orfrom about 3 wt % to 5 wt %). Generally, the acid soluble ash fractionof corn stover constitutes from about 35 to about 45 wt % of the ash andthe acid insoluble ash fraction constitutes from about 55 wt % to about65 wt % of the ash of the feedstock.

Wheat straw typically has an ash content of at least 3 wt % or fromabout 3 wt % to about 10 wt %. Additionally or alternatively, wheatstraw typically has a silica content of at least about 1 wt %, fromabout 1 wt % to about 7 wt % or from about 1 wt % to 5 wt %. Generally,the acid soluble ash fraction of wheat straw constitutes from about 35to about 45 wt % of the ash and the acid insoluble ash fractionconstitutes from about 55 wt % to about 65 wt % of the ash of thefeedstock.

Woody biomass feedstocks generally have an ash content of less thanabout 5 wt % or less than about 2 wt %. Typically, woody biomassfeedstocks have an ash of from about 0.1 wt % to about 5 wt % or moretypically from about 0.1 wt % to about 2 wt %. Generally, woody biomassfeedstocks have a silica content less than about 0.2 wt % or from about0.01 wt % to about 0.2 wt %.

7. Dry Cleaning

As noted, the ash portion of biomass feedstocks utilized in accordancewith the present invention typically includes one or more inorganiccomponents such as, for example, silica, calcium-containing components,magnesium-containing components, sodium-containing components,potassium-containing components, phosphorus-containing components,aluminum-containing components, and combinations thereof. Theseinorganic components may interfere with acid impregnation for thepurpose of preparation of the feedstock for enzymatic hydrolysis toproduce fermentable sugars. More particularly, these components mayreact with and/or neutralize the acid utilized in acid impregnation,thereby rendering at least a portion of the acid contacted with thebiomass feedstock significantly less effective or even ineffective foracid impregnation and preparation of the feedstock for enzymatichydrolysis to produce fermentable sugars. Compensating for the reductionin effectiveness of the acid for impregnation may require additionalacid loading either in the form of a more concentrated acidic liquidmedium and/or utilizing additional acidic liquid medium in the acidimpregnation step. Utilizing a more concentrated acidic liquid medium isgenerally undesired based on handling concerns and/or the increased riskof equipment corrosion. Utilizing additional acidic liquid mediumlikewise raises these concerns and also generation of additional wasteliquid that must be handled and disposed. Thus, a need exists for amethod for providing a biomass feedstock having a significant portion ofthe ash fraction removed, thereby preferably avoiding the need forproviding additional acid either through use of a more concentratedacidic liquid medium and/or use of additional acidic liquid medium.

Inorganic components of ash, such as, for example, silica, may alsonegatively affect process equipment by increasing wear, particularly onmoving parts. Therefore removal of a significant portion of ash may alsobeneficially increase the life of process equipment.

In accordance with the present invention, it has been discovered that asignificant fraction of the ash portion may be removed from the biomassfeedstock by a process in which various fractions are removed from thebiomass feedstock prior to acid impregnation. Generally, removal of ashfrom the biomass feedstock for the purpose of improving theeffectiveness of acid impregnation includes removal of a fineparticulate fraction from the biomass feedstock prior to acidimpregnation. Additionally, the method may include removal of a fractionrich in dense contaminants from the biomass feedstock. This densecontaminant fraction contains various components that are undesiredsince they do not contribute to fermentable sugar and/or ethanol yieldand their presence may inhibit preparation of the feedstock for recoveryof fermentable sugars and recovery of fermentable sugars. Removal ofthese fractions generally proceeds by one or more separation, orclassification techniques. As used herein, the terms “classified,”“classifying,” or “classification” refer to any operation that iscapable of separating the solids component of a biomass feedstock intotwo or more fractions having different particle size ranges. Severalclassification techniques may be used in accordance with the presentinvention. For example, suitable classification techniques include airclassifying, screen separation, filtration, and sedimentation techniquesor by means of a cyclone separator. In various preferred embodiments asdetailed herein, separating the solids component of a biomass feedstockinto two or more fractions having different particle size ranges isconducted by air classification and/or screen separation.

Removal of the ash portion of the biomass feedstock in accordance withthe present invention by a combination of one or more classifying and/orscreening operations generally proceeds in the absence of addingmoisture to the biomass feedstock by, for example, contacting thebiomass feedstock or any fractions removed therefrom with wash water.Accordingly, the methods for removal of the ash fraction from thefeedstock detailed herein may conveniently be referred to as “drycleaning” methods. Avoiding addition of wash water at this stage in theprocess provides many advantages. The fine particulate fraction of thebiomass feedstock is rich in ash content. Water washing methods areknown that remove fine particulates. However, in addition to removal ofthe fine particulate fraction, water washing also dissolves a portion ofsoluble components of the biomass feedstock including, for example,cellulose, hemicellulose, and starches. Removal of these solublecomponents at this point in the process reduces fermentable sugar and/orethanol yields during later processing. In addition, water washing maybe undesired since it may provide a relatively moist feedstock for acidimpregnation, which may be undesired because it may hinder dispersion ofthe acid throughout the feedstock. The dry cleaning methods detailedherein provide removal and recovery of the ash fraction in the form of afine particulate fraction, but without undesired removal of solublecomponents of the biomass feedstock. Dry cleaning ash removal methodsare likewise advantageous since they do not introduce additional liquidloading to the process, which would add to the proportion of wastewaterto be disposed of and/or cleaned prior to use. In addition, as notedabove and detailed elsewhere herein, removal of a fine particulatefraction rich in ash content contributes to improving the effectivenessof acid impregnation.

As noted, the ash component of the biomass feedstock includes an acidsoluble fraction and an acid insoluble fraction. The dry cleaningmethods detailed herein are effective for removal of a fine particulatefraction including both acid soluble and acid insoluble ash componentsand may provide removal of a greater proportion of total ash thanprovided by water washing, but without the concomitant removal ofdesired soluble components and problems associated with use of a moistfeedstock in acid impregnation. However, since wash water is notutilized typically the remaining ash fraction of biomass feedstockscleaned in accordance with the present dry cleaning methods may includea greater proportion of acid soluble ash components than remaining afterwater washing. Thus, the soluble ash fraction of cleaned biomassfeedstocks provided by the present invention may constitute a greaterproportion of the total ash content of cleaned feedstocks provided bywater washing. Thus, cleaned biomass feedstocks provided by the drycleaning methods detailed herein may provide a cleaned biomass feedstockhaving a lower ash content than provided by wet cleaning, but an ashcontent identified by a higher proportion of acid soluble ash componentsrelative to the total ash content of the cleaned biomass feedstockand/or acid insoluble components as compared to water washing methods.In this manner, cleaned biomass feedstocks provided by the presentinvention are identified, or marked by the proportion of soluble ashcomponents of the ash fraction. Although these ash components aresoluble during acid impregnation, such cleaned biomass feedstocks aresuitable for acid impregnation because of the low overall ash content.

FIG. 1A depicts a dry cleaning method of the present invention. As shownin FIG. 1A, biomass feedstock 300 is introduced into a contaminantseparation system 310 for removal of a dense contaminant fraction 315from the biomass feedstock to provide a biomass feedstock 320.Additionally, biomass feedstock 320 is introduced into a fineparticulate separation zone 330 for removal of a fine particulatefraction 335 from the biomass feedstock to provide a cleaned biomassfeedstock 340.

Optionally as shown in FIG. 1A, biomass feedstock 300 may be subjectedto a particle size reduction operation (e.g., milling or grinding) in asuitable vessel or zone (i.e., 305 shown in FIG. 1A) to provide biomassfeedstock 305A for introduction into contaminant separation zone 310.Additionally or alternatively, biomass feedstock 320 exiting thecontaminant separation zone may be subjected to a particle sizereduction operation in a suitable zone or vessel (i.e., 325 shown inFIG. 1A) to provide a biomass feedstock 325A for introduction into fineparticulate separation zone 330.

In various preferred embodiments, the one or more optional particle sizereduction operations provides a biomass feedstock 305A for introductioninto the contaminant separation zone 310 and/or a biomass feedstock 325Afor introduction into the fine particulate separation zone 330 having apreferred particle size distribution. For example, typically biomassfeedstock 305A and/or biomass feedstock 325A is in the form of aparticulate biomass feedstock comprising particles having a particlesize distribution such that no more than from about 20 to about 40 wt %of the feedstock particles are retained on a screen having openings ofabout U.S. Sieve No. 5 (4 mm) In various other preferred embodiments,the biomass feedstock is in the form of a particulate biomass feedstockcomprising particles having a particle size distribution such that fromabout 90 to about 95 wt % of the feedstock particles are retained on ascreen having openings of about U.S. Sieve No. 60 (250 μm).

(i) Contaminant Separation Zone

Again with reference to FIG. 1A, in accordance with various preferredembodiments, contaminant separation zone 310 comprises one or more airclassifier(s) in which the biomass feedstock is contacted with a gasstream (e.g., air) in the air classifier(s). Suitable air classifiersinclude air density separators, cyclone separators, falling bedaspirators, and turbo air classifiers. Operation of the air classifiergenerally proceeds in accordance with methods known in the art anddepending on the composition of the biomass feedstock introduced intothe contaminant separation zone (e.g., the proportion of the feedstockconstituted by the dense contaminant fraction to be removed). Generally,the air velocity within the air classifier is maintained (e.g., byadjusting the damper) such that the dense, or heavy contaminants are notcarried forward, or through the air classifier with the air flow. Invarious embodiments of the present invention, the contaminant separationzone includes a series of air classifiers wherein the biomass feedstockis contacted with the gas stream in each of the series of airclassifiers, thereby forming a plurality of dense contaminant fractionsand a plurality of biomass feedstock fractions depleted in densecontaminants. In these and other embodiments the contaminant separationzone includes at least 2, at least 3, or at least 4 air classifiers.

In various other embodiments, the contaminant separation zone includesat least one classifying screen, thereby recovering the densecontaminant fraction on the at least one classifying screen and thebiomass feedstock depleted in dense contaminants having passed throughthe at least one classifying screen. In these embodiments the at leastone classifying screen of the contaminant separation zone typically hasopenings of a size at least about 0.5 inches (12.7 mm) or from about 0.5inches (12.7 mm) to about 1 inch (25.4 mm) Typically, dense contaminantfraction 315 removed from the biomass feedstock utilizing thecontaminant separation zone comprises one or more components selectedfrom the group consisting of gravel and metal impurities. The densecontaminant fraction also typically contains biomass feedstock particleshaving a particle size distribution such that at least about 95 wt % ofthe particles are retained on a screen having openings of a size ofabout 0.5 inches (12.7 mm)

(ii) Fine Particulate Separation Zone

Again with reference to FIG. 1A, biomass feedstock 320 (or optionally325A) is introduced into a fine particulate separation zone 330 forremoval of a fine particulate fraction 335 and formation of a cleanedbiomass feedstock 340. Generally, fine particulate separation zone 330comprises at least one classifying screen to recover the cleaned biomassfeedstock 340 on the at least one classifying screen and the fineparticulate fraction 335 having passed through the screen. Various typesof screens may be used in the screen classification system including,for example, woven wire screens and/or wedge wire screens.

In various preferred embodiments, the screen separation system includesone screen. Typically, the screen has openings of a size of from aboutU.S. Sieve No. 100 (150 μm) to about U.S. Sieve No. 20 (840 μm) or fromabout U.S. Sieve No. 80 (175 μm) to about U.S. Sieve No. 60 (250 μm).For example, typically the screen separation system comprises a screenhaving openings of a size of about U.S. Sieve No. 20 (840 μm), aboutU.S. Sieve No. 60 (250 μm), about U.S. Sieve No. 80 (175 μm), or aboutU.S. Sieve No. 100 (150 μm).

In various other embodiments, the screen separation system comprises twoscreens. Typically, the first screen has openings of a size of fromabout 0.5 inches to about 1 inch and the second screen has openings of asize of from about U.S. Sieve No. (840 μm) to about U.S. Sieve No. 100(150 μm).

In still further embodiments, the screen separation system comprisesthree screens or four screens including, for example, one or morescreens having openings of a size of from about 0.5 inches to about 1inch and arranged, for example, in series and one or more screens havingopenings of a size of from about U.S. Sieve No. (840 μm) to about U.S.Sieve No. 100 (150 μm) arranged, for example, in series.

In various preferred embodiments the contaminant separation zone andfine particulate separation zone are combined in a single zone thatincludes one or more air classifiers equipped with one or moreclassifying screens. Accordingly, in these embodiments, utilizing one ormore air classifiers further comprising one or more classifying screensprovides for the recovery of (i) a dense contaminant fraction retainedon the at least one classifying screen, (ii) a cleaned biomass feedstockhaving passed through the at least one classifying screen, and (iii) afine particulate fraction having been entrained in the gas streamexiting the air classifier. In various embodiments, the at least oneclassifying screen of the air classifier has openings of a size at leastabout 0.5 inches (12.7 mm) or from about 0.5 inches (12 7 mm) to about 1inch (25.4 mm)

The contaminant separation zone and/or fine particulate separation zonemay include a magnetic separation system to remove a ferromagneticfraction from the biomass feedstock prior to removal of the fineparticulate fraction therefrom and/or biomass feedstock depleted indense contaminants. The location or position of the magnetic separationsystem is not narrowly critical and may be placed at any point in thecleaning process that is effective for removal of such ferromagneticfraction.

(iii) Fine Particulate Fraction

As noted, it is currently believed that a significant fraction of theash portion of the biomass feedstock is present in the form ofrelatively small particulates. Since the ash portion of the biomassfeedstock contains various components that impede or interfere with acidimpregnation, removal of a fine particulate fraction and the concomitantremoval of a significant portion of the ash fraction of the biomass isadvantageous since it contributes to improved effectiveness of the acidimpregnation. In accordance with the present invention it has beendiscovered that recovering a fine particulate fraction comprisingparticles within various preferred size ranges provides for advantageousremoval of a significant fraction of the ash portion of the biomassfeedstock present in the fines of the biomass feedstock. For example, inaccordance with various preferred embodiments, at least about 95 wt % ofthe fine particulate fraction passes through a screen having openingsfrom about U.S. Sieve No. 100 (150 μm) to about U.S. Sieve No. 20 (840μm). In various other embodiments, at least about 95 wt % of the fineparticulate fraction passes through a screen having openings from aboutU.S. Sieve No. 80 (175 μm) to about U.S. Sieve No. 60 (250 μm). In stillfurther preferred embodiments, at least about 95 wt % of the fineparticulate fraction passes through a screen having openings of aboutU.S. Sieve No. 60 (250 μm), through a screen having openings of aboutU.S. Sieve No. 80 (175 μm), or through a screen having openings of aboutU.S. Sieve No. 100 (150 μm).

In addition to the particle size distribution, ash content of the fineparticulate fraction may also indicate effective cleaning of thefeedstock. Since a significant fraction of the ash content of thebiomass feedstock is present in fine particulates, the fine particulatefraction typically has significantly higher ash content (i.e., weightpercent ash) than the biomass feedstock introduced into the particulatesize separation system 330. Generally, the fine particulate fraction hasan ash content of at least about 30 wt %, at least about 40 wt %, or atleast about 50 wt %. Typically, the fine particulate fraction has an ashcontent of from about 30 wt % to about 80 wt %, more typically fromabout 40 to about 70 wt % and, still more typically from about 45 toabout 60 wt %. Typically, the fine particulate fraction has a silicacontent of at least about 30 wt %, at least about 40 wt %, or from about30 wt % to about 50 wt %, or from about 40 wt % to about 50 wt %.

Generally, an acid soluble ash fraction constitutes from about 20 wt %to about 40 wt % of the ash of the fine particulate fraction. Typically,potassium constitutes at least about 30 wt % or from about 35 wt % toabout 45 wt % of the acid soluble fraction. An acid insoluble fractiongenerally constitutes from about 60 wt % to about 80 wt % of the ash ofthe fine particulate fraction. Typically, silica constitutes at leastabout 90 wt % or at least about 95 wt % (e.g., from about 95 to about 99wt %) of the acid insoluble fraction.

In addition to the absolute proportion of ash content of the fineparticulate fraction, the proportion of ash present in the fineparticulate fraction relative to the ash content of the biomassfeedstock is an indicator of effective ash removal. For example,generally the ratio of the ash content (wt % ash) of the fineparticulate fraction to the ash content of biomass feedstock is at leastabout 3:1, at least about 4:1, at least about 5:1, at least about 6:1,at least about 7:1, or at least about 8:1. For example, in variouspreferred embodiments in which the ash content of the biomass feedstockis from about 8 wt % to about 12 wt %, the ash content of the fineparticulate fraction is at least about 40 wt %, at least about 50 wt %,at least about 60 wt %, from about 40 wt % to about 90 wt %, from about40 wt % to about 80 wt %, or from about 50 wt % to about 80 wt %.

Generally, the fine particulate fraction has a moisture content of lessthan about 20 wt %, less than about 15 wt %, or less than about 10 wt %.Typically, the fine particulate fraction has a moisture content of fromabout 1 wt % to about 20 wt %, more typically from about 1 to about 15wt % and, still more typically, from about 1 wt % to about 10 wt %.Generally, the fine particulate fraction has a cellulose content of fromabout 10 wt % to about 40 wt %, a hemicellulose content of from about 10wt % to about 30 wt %, and/or a lignin content of from about 5 wt % toabout 25 wt %.

(iv) Cleaned Biomass Feedstock

As noted, removal of various fractions from the biomass feedstock inaccordance with the present invention as depicted in FIG. 1A provides acleaned biomass feedstock having various contaminants removed therefromincluding, for example, various dense contaminants and a fineparticulate fraction rich in ash content.

Generally, the cleaned biomass feedstock has an ash content of less thanabout 10 wt %, less than about 9 wt %, less than about 8 wt %, less thanabout 7 wt %, less than about 6 wt %, less than about 5 wt %, less thanabout 4 wt %, less than about 3 wt %, less than about 2 wt %, or lessthan about 1 wt %. For example, typically the ash content of the cleanedbiomass feedstock is from about 0.1 wt % to about 10 wt %, from about0.1 wt % to about 8 wt %, or from about 0.1 wt % to about 7 wt %, fromabout 0.1 wt % to about 6 wt %, or from about 0.1 wt % to about 5 wt %.

In addition to a low proportion of total ash content, removal of asignificant fraction of the ash component of the biomass feedstock mayalso be indicated by the proportion of ash content of the cleanedbiomass feedstock as compared to the starting ash content of thefeedstock. For example, in various embodiments, the ash content of thecleaned biomass feedstock (i.e. , wt % ash) is no more than about 75%,no more than about 70%, no more than about 65%, no more than about 60%,no more than about 55%,of the ash content of the biomass feedstock (dryweight basis). Typically, the ash content of the cleaned biomassfeedstock is no more than about 50%, no more than about 45%, or no morethan about 40% of the ash content of the biomass feedstock (dry weightbasis).

As noted, the ash portion of the biomass feedstock and fractions removedtherefrom includes soluble and insoluble components. Generally, the ashcontent of the cleaned biomass feedstock indicates removal of asignificant fraction of the ash of the biomass feedstock. However, ascompared to water washing methods, the ash content of the cleanedbiomass feedstock provided by the present dry cleaning methods typicallyincludes a higher fraction of acid soluble ash components. For example,typically the acid soluble ash fraction constitutes at least about 30 wt%, at least about 35 wt %, or at least about 40 wt % (e.g., from about35 wt % to about 45 wt % or from about 40 wt % to about 45 wt %) of thecleaned biomass feedstock ash content. Retention of an acid soluble ashfraction in cleaned biomass feedstocks in accordance with the presentinvention thus represents removal of undesired ash from the biomassfeedstock, but without the attendant disadvantages of water washing(e.g., removal of other desired soluble components of the biomassfeedstock such as cellulose, hemicellulose, and other starches).

Effective cleaning methods may also be indicated by the relativeproportions of the ash content of the fine particulate fraction andcleaned biomass feedstock. For example, typically the ratio of the ashcontent of the fine particulate fraction (wt % ash) to the ash contentof the cleaned biomass feedstock is at least about 3:1, at least about4:1, or at least about 5:1. In accordance with various preferredembodiments, the ratio of the ash content of the fine particulatefraction (wt % ash) to the ash content of the cleaned biomass feedstockis at least about 6:1, at least about 7:1, at least about 8:1, at leastabout 9:1, at least about 10:1, at least about 11:1, at least about12:1, at least about 13:1, at least about 14:1, or at least about 15:1.

Generally, the cleaned biomass feedstock has a moisture content of lessthan about 20 wt %, less than about 15 wt %, or less than about 10 wt %.Typically, the cleaned biomass feedstock has a moisture content of fromabout 1 wt % to about 20 wt %, more typically from about 3 to about 15wt % and, still more typically, from about 5 wt % to about 10 wt %. Asnoted, the dry cleaning methods detailed herein are advantageouslyconducted in the absence of addition of liquid, or wash water for thepurpose of removing contaminants. Accordingly, the moisture content ofthe feedstock and fractions removed therefrom are relatively constant.For example, generally the moisture content of one or more andpreferably each of the biomass feedstock, dense contaminant fraction,biomass feedstock depleted in dense contaminants, cleaned biomassfeedstock, and fine particulate fraction vary by no more than about 10wt %, no more than about 5 wt %, or no more than about 3 wt %.Typically, the moisture content of one or more and preferably each ofthe biomass feedstock, dense contaminant fraction, biomass feedstockdepleted in dense contaminants, cleaned biomass feedstock, and fineparticulate fraction vary by no more than about 1 wt %.

Generally, the cleaned biomass feedstock has a cellulose content of atleast about 30 wt % or from about 30 wt % to about 60 wt %. Generally,the cleaned biomass feedstock has a hemicellulose content of at leastabout 20 wt % or from about 20 wt % to about 40 wt %. Generally, cleanedbiomass feedstock has a lignin content of at least about 10 wt % or fromabout 10 wt % to about 25 wt %.

Typically, the cleaned biomass feedstock has a particle sizedistribution such that at least about 95 wt % of the particles areretained on a screen having openings of a size of about U.S. Sieve No.60 (250 μm). Typically, the cleaned biomass feedstock has a particlesize distribution such that from about 95 to about 99 wt % of thebiomass feedstock is retained on a screen having openings of about U.S.Sieve No. 60 (250 μm).

8. Power Requirements

Preferably, acid impregnation is conducted in a manner that balanceseffective impregnation with power consumption, and the costs associatedtherewith. It is currently believed that many factors affect therequired power to provide effective acid impregnation including, forexample, the composition of the feedstock and/or acidic liquid medium.Additionally or alternatively, the conditions of contact including, forexample, contact time, contact temperature, and manner of contact (e.g.,by spraying or soaking). Generally in accordance with the presentinvention (and as detailed in Example 2), it has been observed thatpower inputs of less than about 10 kilowatt hours per ton corn stover(kWh/ton), or less than about 8 kWh/ton provide effective acidimpregnation. Typically, the power input ranges from about 1 to about 8kWh/ton or from about 3 to about 6 kWh/ton.

9. Acid-Impregnated Feedstock

Contact of the feedstock and acidic liquid medium generally provides anacid-impregnated feedstock (33 in FIG. 1) in the form of a slurry orcake of particulate biomass dispersed throughout a liquid medium.

Relatively low moisture content of the acid-impregnated feedstockgenerally reduces the energy required during subsequent heating.Accordingly, in various preferred embodiments the acid-impregnatedfeedstock generally has a moisture content of less than about 70 wt %,typically less than about 60 wt %, more typically less than about 55 wt% and, still more typically, less than about 50 wt %. Such moisturecontents may be achieved by spraying an acidic liquid medium onto thefeedstock, soaking the feedstock in an appropriate proportion of acidicliquid medium and/or soaking the feedstock followed by dewatering.

In addition to promoting dispersion of the acid throughout thefeedstock, a certain proportion of moisture in the acid-impregnatedfeedstock is preferred in order to significantly minimize, andpreferably substantially avoid the risk of pyrolysis of the feedstockduring subsequent treatment at elevated temperature and pressure (e.g.,steam treatment as detailed elsewhere herein). Thus, typically themoisture content of the acid-impregnated feedstock is at least about 20wt %, more typically at least about 30 wt % and, still more typically,at least about 40 wt %.

Accordingly, in various preferred embodiments, the moisture content ofthe slurry is typically from about 20 wt % to about 70 wt % or fromabout 30 wt % to about 60 wt %, preferably from about 35 wt % to about55 wt % and, more preferably, from about 40 wt % to about 50 wt %.

Regardless of the manner of contact of the feedstock and acid (e.g.,soaking, soaking followed by dewatering, or spraying), generally theacid-impregnated biomass slurry exhibits a solids content of at leastabout 0.25 g solids per g slurry. Typically, the acid-impregnatedfeedstock exhibits a solids content of at least about 0.30 g solids perg slurry and, more typically, at least about 0.35 g solids per g slurry.For example, in various embodiments, the acid impregnated biomass slurryexhibits a total solids content of from about 0.35 to about 0.65 gsolids per g wet mixture, or from about 0.45 to about 0.55 g solids perg wet mixture.

10. Washing

In addition to impregnation of the feedstock to facilitate furthertreatment, acid impregnation may be utilized in a method that removesone or more impurities from the feedstock. For example, field-harvestedfeedstock may comprise one or more impurities (e.g., ash, sand, soil,rock, and tramp metals). In addition, contact of the feedstock and anacidic liquid medium may generate one or more impurities such as, forexample, phenolic compounds derived from the lignin portion of thecomplex. These impurities may inhibit subsequent enzymatic hydrolysis ofpretreated feedstock. Accordingly, in various preferred embodiments,acid-impregnated feedstock is subjected to a washing operation (notshown in FIG. 1) prior to further processing. More particularly, anaqueous liquid fraction may typically be removed from theacid-impregnated feedstock to form an acid-impregnated feedstock havinga reduced content of one or more impurities.

Generally, washing of the feedstock may be conducted by counter-currentcontact of the feedstock and a liquid washing medium. Typically, thewashing medium is an aqueous medium (e.g., process water) and thewashing is conducted utilizing a suitable vessel or reactor known in theart.

The goal of washing is removal of impurities that may impact furtherprocessing. However, preferably the washing operation does not degradethe biomass feedstock fibers to a degree that substantially inhibits orprevents deriving fermentable sugars from the feedstock. Generally, thesolid phase biomass comprises a fibrous solid phase that may becountercurrently contacted with the washing liquid in a mixing zone. Invarious preferred embodiments, the mixing zone comprisescounter-rotating shafts (generally in parallel arrangement) havingflights for agitation of the biomass. The conditions of countercurrentcontact and agitation of the biomass are controlled to provide contactthat increases the bioavailability of cellulose without excessivedegradation of the fibers of the solid phase. For example, typically thewashing does not degrade the fibers by more than 5%, more typically nomore than about 3% and, still more typically, no more than about 2%, asmeasured by the average length of the fibers after the washing ascompared to the average length of the fibers before washing.

FIG. 2 depicts a method that combines washing and dilute acidimpregnation of biomass. As shown in FIG. 2, milled and cleanedfeedstock 200 (i.e., 21 shown in connection with the process of FIG. 1)is introduced into vessel 205 to which is also introduced acid stream210. The milled/cleaned feedstock and acid are typically contactedwithin the vessel at a temperature of from about 20° C. to about 90° C.and, more typically, from about 30° C. to about 80° C. The contact timewithin vessel 205 is typically from about 0.1 to about 20 minutes and,more typically, from about 0.5 to about 10 minutes. Generally, theproportion of acidic liquid medium introduced into vessel 205 issuitable to provide a slurry in the vessel having a solids concentrationin the vessel of from about 4 to about 10 wt %. To loosen impuritiessuch as sand and soil from the feedstock, typically the slurry isagitated using a suitable agitator or mixer known in the art.

Again with reference to FIG. 2, acid-impregnated feedstock 215 isremoved from vessel 205 and introduced into vessel 220 and held withinthe vessel for a time that promotes dispersion of acid throughout theparticles of the acid-impregnated feedstock and to allow heavycontaminants such as gravel, sand, and metal to disengage from thefeedstock fibers and settle to the bottom of the vessel. Generally, theacid-impregnated feedstock is held in vessel 220 for from about 1 toabout 60 minutes, or from about 1 to about 30 minutes. The temperatureof the feedstock within vessel 220 is typically from about 20° C. toabout 90° C. and, more typically, from about 30° C. to about 80° C. Asthe solids content of the acid-impregnated feedstock decreases,solubilization of sugars may increase, which is undesired since this mayreduce the ultimate fermentable sugar yield. If necessary, the solidscontent of the acid-impregnated feedstock may be controlled bycontrolling the amount of acid solution added to the feedstock and/orremoval of excess acid solution using solid/liquid separators.Preferably, the solids content of the acid-impregnated biomass feedstockis from about 5 wt % to about 15 wt %, more preferably from about 5 toabout 10 wt % and, still more preferably, from about 5 wt % to about 7wt %.

Again with reference to FIG. 2, acid-impregnated feedstock 215A isintroduced into a solids/liquid separator 225 comprising a drainingscrew conveyor, screen, filter, centrifuge, settler, dewatering screwpress, or other solid/liquid separation instrumentality for removal ofliquid (e.g., water). The manner of liquid removal is not narrowlycritical and generally proceeds in accordance with suitable methodsknown in the art. Removal of liquid from the acid-impregnated feedstockprovides acid-impregnated feedstock 230. It is to be understood that theacid-impregnated feedstock of the process of FIG. 1 (i.e., 33 in FIG. 1)may be prepared by the process depicted in FIG. 2. Contact of thefeedstock and acid may proceed by soaking of the feedstock in an acidicliquid medium and/or spraying of the acidic liquid medium onto thefeedstock, as detailed elsewhere herein. Acidic washing is generallybelieved to promote distribution of acid throughout the biomassparticles while also removing a portion (preferably a significantportion) of the contaminants present in the feedstock (e.g., dirt, sand,and gravel). Removal of contaminants is advantageous as these mayinterfere or impact downstream processing by, for example, wear and/ordamage to equipment utilized.

Again with reference to FIG. 2, dewatering acid-contacted feedstock 215Aproduces aqueous waste stream 235. In addition to promoting dispersionof the acid throughout the feedstock particles, holding acid-contactedfeedstock 215 within vessel 220 allows for removal of impurities fromthe acid-washed feedstock and removal of water from the feedstock thatcould interfere with subsequent dewatering of the feedstock. Inaccordance with the process depicted in FIG. 2, vessel 220 is equippedwith suitable apparatus (e.g., a screen) by or through which variousfine particulate impurities are removed from the acid-washed feedstock.Further in accordance with the process of FIG. 2, impurities 240 areremoved from vessel 220 and combined with aqueous waste stream 235 andintroduced into another solid/liquid separator 245, e.g., a screen,filter, centrifuge, settler, hydrocyclone or flotation vessel. Alsointroduced into solid/liquid separator 245 is water stream 250.Separator 245 may be equipped for recovery of a variety of relativelyfine impurities that have been separated from the acid-washed feedstock.The manner of recovery of fraction 255 from separator 245 is notnarrowly critical and advantageously may be accomplished byincorporating a screen sized for recovery of the desired particles. Inaccordance with the process depicted in FIG. 2, impurities 255 are inthe form of an sand-rich product (e.g., an acid-washed sand fraction).Recovery of fraction 255 from separator 245 yields a spent acid stream260 in the form of an acidic aqueous liquid fraction. As shown in FIG.2, spent acid stream 260 is recycled and combined with fresh acid 265 invessel 270 to form a treatment acid stream 272 that is heated in vessel275 to form heated treatment acid stream 210 that is contacted with thecleaned/milled feedstock. Although shown in FIG. 2, recovery of animpurity fraction (e.g., acid-washed sand) is not required. That is, theprocess of FIG. 2 may be utilized simply for the purpose of acidimpregnation and removal of impurities from the feedstock as describedabove.

(i) Neutralization Capacity

Without being bound by theory, it is currently believed that during acidimpregnation, typically only a portion of the acidic liquid mediumcontributes in any significant degree to preparation of the feedstockfor enzymatic hydrolysis to produce fermentable sugars. Fineparticulates of the biomass feedstock are generally high in inorganicscomponents, including, for example, silica, calcium-containingcomponents, magnesium-containing components, sodium-containingcomponents, potassium-containing components, phosphorus-containingcomponents, aluminum-containing components, and combinations thereofThese components of the biomass feedstock may neutralize the acid duringacid impregnation. Consumption or neutralization of a significantfraction of the acidic liquid medium by the fine particulate fraction(e.g., as a result of the presence of alkaline compounds) is undesiredas it generally does not provide acid impregnation that contributes inany significant degree to preparation of the feedstock for enzymatichydrolysis to produce fermentable sugars. Various methods for cleaningthe biomass feedstock detailed above (e.g., dry cleaning) remove asignificant portion of the fine particulate fraction of the biomassfeedstock, in particular, a fine particulate fraction rich in ashcontent.

The presence of components that may neutralize the acid during acidimpregnation may be indicated by an acid neutralization capacity of thefeedstock determined in accordance with methods known in the art. Inparticular, the acid neutralization capacity of biomass feedstocks andfractions removed and derived therefrom (e.g., fine particulatefractions and cleaned biomass feedstocks) may be determined inaccordance with Protocol A as detailed in Example 8. Preferably inaccordance with the present invention, the dry cleaning methods detailedherein provide cleaned biomass feedstocks having a lower acidneutralization capacity than achieved by other methods (e.g., waterwashing). For example, typically cleaned biomass feedstocks prepared inaccordance with the present invention have an acid neutralizationcapacity as determined in accordance with Protocol A of less than about0.01, more typically less than about 0.009 and still more typically,less than about 0.008, or less than about 0.007 (grams of acid/gram ofdry biomass). In various embodiments, cleaned biomass feedstocks have anacid neutralization capacity of from about 0.0001 to about 0.01, fromabout 0.001 to about 0.01, from about 0.002 to about 0.01, from about0.003 to about 0.01, from about 0.0001 to about 0.009, or from about0.0001 to about 0.008.

Additionally or alternatively, an effective cleaning method may beindicated by a reduced acid neutralization capacity of the cleanedfeedstock as compared to the starting biomass feedstock. For example,typically the acid neutralization capacity of the cleaned particulatebiomass feedstock is no more than about 90% of the acid neutralizationcapacity of the starting feedstock, no more than about 85% of the acidneutralization of the starting feedstock, no more than about 80% of theacid neutralization capacity of the starting feedstock, no more thanabout 75%, no more than about 70%, no more than 65% no more than about60%, no more than about 55%, no more than about 50%, or no more thanabout 45% of the acid neutralization capacity of the biomass feedstock.In various embodiments, the acid neutralization capacity of the cleanedparticulate biomass feedstock is from about 10% to about 95%, from about10% to about 90%, from about 10% to about 80%, from about 20% to about95%, from about 20% to about 90%, from about 20% to about 80%, fromabout 30% to about 95%, from about 30% to about 90%, from about 30% toabout 80%, from about 40% to about 95%, from about 40% to about 90%,from about 40% to about 80%, from about 50% to about 95%, from about 50%to about 90%, from about 50% to about 80%, from about 60% to about 95%,from about 60% to about 90%, from about 60% to about 80%, from about 70%to about 95%, from about 70% to about 90%, or from about 70% to about80%,of the acid neutralization capacity of the biomass feedstock.Effective cleaning methods may be indicated by either or both thesemeasures of acid neutralization capacity. That is, cleaned biomassfeedstocks may exhibit an acid neutralization capacity within theabove-noted limits and ranges and/or that exhibit a reduction in acidneutralization capacity as compared to the acid neutralization capacityof the starting feedstock.

Additionally or alternatively, effective cleaning methods may also beindicated by the acid neutralization capacity of the fine particulatefraction. For example, typically the fine particulate fraction has anacid neutralization capacity as determined in accordance with Protocol Aof at least about 0.001, at least about 0.005, at least about 0.008, atleast about 0.01, at least about 0.012, at least about 0.02, at leastabout 0.03, at least about 0.04, or at least about 0.05. Since asignificant fraction of the neutralizing components of the ash of thefeedstock are present in the fine particulate fraction, the relativeacid neutralization capacities of fine particulate fraction and thecleaned biomass feedstock and/or starting biomass feedstock may alsoindicate an effective cleaning method. For example, typically the ratioof the acid neutralization capacity of the fine particulate to the acidneutralization capacity of the cleaned biomass feedstock is at leastabout 0.5:1, at least about 0.7:1, at least about 0.9:1, at least about1.1:1, at least about 1.3:1, at least about 1.5:1, or at least about2:1.

(ii) Acid Consumption

Effective cleaning methods of the present invention may also beindicated by the effectiveness of the acid consumed by the feedstockduring acid impregnation. In particular, effective acid consumption maybe indicated by the effectiveness of acid impregnation for derivingfermentable sugars (e.g., hemicellulose-derived sugars) and/or providinga feedstock that during subsequent enzymatic hydrolysis for the purposeof deriving fermentable sugars provides advantageous fermentable sugaryields. Derivation of xylose during pretreatment and providing apretreated feedstock having a cellulose digestibility effective forsubsequent enzymatic hydrolysis may be determined in accordance withmethods known in the art including, for example, as described inExamples 10 and 11, respectively. For example, effective cleaningmethods may be indicated by either or both of these measures incombination with use of a relatively low proportion of acid during acidimpregnation. That is, since the feedstock has been cleaned and asignificant fraction of the feedstock that consumes the acid but doesnot contribute to effective acid impregnation has been removed, loweramounts of acid (e.g., a relatively proportion of acid diluted in aliquid medium to the total proportion of biomass feedstock solids) maybe utilized during acid impregnation that nonetheless provide effectiveacid impregnation.

For example, in accordance with various preferred embodiments, theweight ratio of acid (i.e., mass of acid) to solids fraction of thecleaned particulate biomass feedstock introduced into the acidimpregnation zone is less than about 0.1:1, less than about 0.05:1, lessthan about 0.045:1, less than about 0.04:1, less than about 0.035:1,less than about 0.03:1, less than about 0.025:1 (e.g., less than about0.02:1 or less than about 0.01:1).

In various embodiments, the weight ratio of acid to solids fraction ofthe cleaned particulate biomass feedstock introduced into the acidimpregnation zone is from about 0.01:1 to about 0.05:1, from about0.01:1 to about 0.045:1, from about 0.01:1 to about 0.04:1, from about0.02:1 to about 0.04:1, from about 0.02:1 to about 0.04:1, from about0.01:1 about 0.035:1, from about 0.02:1 to about 0.035:1, from about0.01:1 to about 0.03:1, from about 0.02:1 to about 0.03:1, from about0.01:1 to about 0.025:1, or from about 0.01:1 to about 0.02:1.Advantageously, as determined in accordance with Protocol B as describedin Example 10, the xylose content of the liquid fraction of a pretreatedbiomass feedstock represents a xylose yield of at least about 70%, atleast about 75%, at least about 80%, at least about 85%, or at leastabout 90% (based hemicellulose content of the particulate biomassfeedstock). In these and other embodiments, the liquid fraction of thepretreated biomass feedstock prepared from the acid-impregnatedfeedstock has a xylose content representing a xylose yield of from about70% to about 95%, from about 70% to about 90%, from about 70% to about85%, from about 75% to about 95%, from about 75% to about 90%, fromabout 75% to about 85%, from about 80% to about 95%, from about 80% toabout 95% or from about 85% to about 95% (based hemicellulose content ofthe particulate biomass feedstock).

Additionally or alternatively, acid impregnated feedstocks provided by arelatively low proportion of acid to biomass solids (e.g., a ratio ofless than about 0.01:1) may be utilized in a process that provides acellulose-containing solids fraction indicating advantageous cellulosedigestibility and thus a pretreated feedstock suitable for effectiveenzymatic hydrolysis. For example, in various preferred embodiments, thecellulose digestibility of the pretreated biomass feedstock, asdetermined in accordance with Protocol C as described in Example 11, isat least about 60%, at least about 70%, at least about 80%, or at leastabout 90%. Typically, the cellulose-derived fermentable sugar content ofthe cellulose hydrolyzate slurry represents a yield of from about 60% toabout 95%, from about 60% to about 90% from about 70% to about 95%; fromabout 70% to about 90%, from about 80% to about 95%, from about 80% toabout 90%, from about 85% to about 95%, or from about 90% to about 95%.

Effective acid impregnation in combination with the dry cleaning methodsmay be provided under various combinations of amount of acid utilized,amount of acidic liquid medium utilized, solids content of the biomassfeedstock, etc.

The acid content of the acidic liquid medium is not narrowly criticaland the desired amount of acid contacted with the cellulosic biomassfeedstock may be controlled by the total amount of acidic liquid mediumcontacted with the biomass feedstock. For example, at higher acidconcentrations, less acidic liquid medium may be utilized. However,generally the acidic liquid medium contacted with the biomass feedstockhas an acid concentration of less than about 5 wt %, less than about 4wt %, less than about 3 wt %, less than about 2 wt %, less than about 1wt %, less than about 0.75 wt %, or less than about 0.5 wt %. Typically,the acidic liquid medium has an acid concentration from about 0.1 wt %to about 4 wt %, from about 0.1 wt % to about 3 wt %, from about 0.2 wt% to about 4.5 wt %, from about 0.5 wt % to about 2 wt %, or from about0.5 wt % to about 1 wt %, from about 0.7 wt % to about 3.5 wt %, fromabout 0.5 wt % to about 3 wt %, from about 1.0 wt % to about 3.0 wt %,or from about 2.0 wt % to about 2.5 wt %.

Generally, the cellulosic biomass feedstock and acidic liquid medium arecontacted at a temperature of at least about 75° C., at least about 100°C., at least about 125° C., at least about 150° C., at least about 175°C. or at least about 200° C. Typically, the cellulosic biomass feedstockand acidic liquid medium are contacted at a temperature of from about100° C. to about 200° C. and more typically from about 125° C. to about175° C.

Although not narrowly critical and generally selected to providesuitable acid uptake, generally the biomass feedstock and acidic liquidmedium are generally contacted for a time of at least about 1 minute, atleast about 2 minutes, at least about 3 minutes, at least about 5minutes, at least about 10 minutes, or at least about 15 minutes.Typically, the biomass feedstock and acidic liquid medium are contactedfor a time of from about 1 minute to about 120 minutes, from about 1minute to about 60 minutes, from about 2 minutes to about 30 minutes,from about 5 minutes to about 30 minutes, or from about 10 minutes toabout 20 minutes.

Further in accordance with these and various other embodiments,utilizing a relatively low proportion of acid may represent utilizing arelatively low proportion of acidic liquid medium. Thus, typically theacid-impregnated biomass feedstock has a total solids content greaterthan typically provided by various conventional methods that includesoaking of the biomass feedstock in a large amount of acidic liquidmedium. Generally, the total solids content of the acid-impregnatedfeedstock is at least about 25 wt %, at least about 30 wt %, at leastabout 40 wt %, or at least about 50 wt %, at least about 60 wt %, atleast about 70 wt %, and at least about 80 wt %. Typically, the totalsolids content of the acid-impregnated biomass feedstock is from about25 wt % to about 90 wt %, from about 25 wt % to about 80 wt %, fromabout 30 wt % to about 90 wt %, from about 30 wt % to about 80 wt %,from about 40 wt % to about 90 wt %, from about 40 wt % to about 80 wt%, from about 50 wt % to about 90 wt %, from about 50 wt % to about 80wt %, from about 60 wt % to about 90 wt %, or from about 60 wt % toabout 80 wt %.

Advantageously, suitable acid uptake is achieved for high solids biomassfeedstocks while utilizing relatively dilute acids. For example,typically the acid concentration of the acidic liquid medium is lessthan about 4 wt %, less than about 3 wt %, or less than about 2 wt %.Additionally or alternatively, suitable acid uptake may be achieved bycontacting relatively low proportions of dilute acidic liquid media. Forexample, the weight ratio of acidic liquid medium to solids content ofthe biomass feedstock contacted in the acid-impregnation vessel is nomore than about 3:1, no more than about 2:1, no more than about 1:1, orno more that about 0.5:1. In various embodiments, the weight ratio ofacidic liquid medium to solids content of the biomass feedstockcontacted in the acid-impregnation vessel is from about 0.5:1 to about4:1, from about 0.5:1 to about 3:1, from 1:1 to about 3:1, from about0.5:1 to about 2:1. Accordingly, in various embodiments, the weightratio of acidic liquid medium to solids content of the biomass feedstockintroduced into the acid-impregnation zone is no more than about 1.1:1and the acidic liquid medium has an acid concentration of less thanabout 4 wt %.

Generally, these embodiments provide an acid-impregnated feedstockhaving a relatively high solids content and a relatively low acidconcentration. For example, in various embodiments, the solids contentof the acid-impregnated feedstock is at least about 25 wt % and the acidconcentration of the acid-impregnated feedstock is less than about 4 wt%.

In various embodiments, an acid-impregnated biomass feedstock having atotal solids content of at least 20 wt % is formed by contacting abiomass feedstock having a total solids content of at least about 80 wt% with an acidic liquid medium having an acid concentration of less thanabout 4 wt % in an acid-impregnation zone, wherein the weight ratio ofacid to solids of the biomass feedstock is no more than about 0.04:1.

B. Steam Treatment

Pretreatment of cellulosic biomass feedstock typically comprisessubjecting the acid-impregnated feedstock to conditions comprisingelevated temperature and pressure to break down thecellulose-hemicellulose-lignin complex. Generally, and again withreference to FIG. 1, acid-impregnated feedstock 33 is subjected toelevated steam pressure and temperature in the presence of H₂O (e.g.,steam) in a suitable reactor, or vessel 37 as shown in FIG. 1. After aperiod of contact with H₂O under the elevated steam pressure andtemperature conditions, the pretreated feedstock is discharged to anenvironment of reduced pressure. The abrupt change in pressure breaksdown the biomass fiber structure including, for example, thecellulose-hemicellulose-lignin complex (e.g., breaks bonds betweenlignin and hemicellulose and/or cellulose).

Steam treatment typically dissociates cellulose from the hemicelluloseand lignin and, thus, provides cellulose available for enzymatichydrolysis to produce fermentable sugars. Steam treatment also typicallydissociates hemicellulose from the complex, generally in the form ofhemicellulose solubilized within a liquid phase of the treatedfeedstock. For example, in various embodiments, at least about 60 wt %,at least about 70 wt %, at least about 80 wt %, or up to 90 wt % of thehemicellulose is solubilized within a liquid phase of the treatedfeedstock. In this manner, steam treatment provides hemicelluloseavailable for hydrolysis to produce fermentable sugars. As describedherein, treatment of acid-impregnated feedstock at elevated temperatureand pressure generally corresponds to treatment known in the artcommonly referred to as “steam explosion.” Steam explosion methods aregenerally described in, for example, U.S. Pat. No. 1,824,221, U.S. Pat.No. 4,461,648, and Canadian Patent No. 1 096 374, the entire contents ofwhich are incorporated herein by reference for all relevant purposes.

As shown in FIG. 1, acid-impregnated feedstock 33 is generallyintroduced into a vessel 37 comprising a contact zone for steamtreatment. The acid-impregnated feedstock is typically in the form of aslurry, or cake. For example, an acid-impregnated slurry may be pressedto form a cake, or plug of pretreated solids for introduction into thesteam treatment vessel. The precise form and configuration of the vesselis not narrowly critical and may be selected by one skilled in the artdepending on the particular circumstances (e.g., properties of thefeedstock and operating conditions). Generally, the vessel includes aninlet for introduction of the feedstock and one or more outlets, orexits for releasing treated feedstock and/or various componentsgenerated during the steam treatment. Once the feedstock is contained inthe vessel, the vessel is pressurized and the feedstock heated.Typically, the feedstock is maintained at a target temperature for atime sufficient to provide suitable heating of the feedstock. After aperiod of pressurizing the vessel and heating the feedstock, thefeedstock is released from the vessel, or contact zone. As noted, theabrupt decrease in pressure during this release promotes break down ofthe cellulose-hemicellulose-lignin complex. That is, the abrupt decreasein pressure causes an explosive effect by virtue of a rapid increase involume of the steam and gases trapped inside the biomass pore structurethat causes high instantaneous incident gas velocities and/or instantvaporization of heated water that has either occupied or been forcedinto the fiber structure so that it becomes literally explosive when itexpands.

Generally, the proportion of steam utilized depends on the initialmoisture content, temperature, and/or void volume of the feedstock.Typically, the ratio of the total mass of H₂O (i.e., steam) toacid-impregnated corn stover introduced into the vessel and/or contactzone is at least about 0.1:1, more typically at least about 0.15:1 and,still more typically, at least about 0.2:1. For example, in variouspreferred embodiments, the mass ratio of H₂O to acid-impregnatedfeedstock is from about 0.1:1 to about 0.5:1 and, more preferably, fromabout 0.2:1 to about 0.4:1, resulting in condensation ofsuperatmospheric water vapor which intermingles with and penetrates thefiber.

1. Pressure

Generally, and in accordance with the process depicted in FIG. 1, steam(typically saturated) 39 is introduced into vessel 37 under a pressureof at least about 75 psig, at least about 125 psig, or at least about150 psig. Typically, acid-impregnated feedstock and H₂O are contactedwithin the vessel (e.g., within a contact zone comprising an inletcomprising a receiving zone for pretreated feedstock and an outlet forremoval of feedstock from the contact zone) under a pressure of fromabout 75 to about 250 psig, more typically from about 90 to about 210psig and, still more typically, from about 150 to about 200 psig.Additionally or alternatively, the acid-impregnated feedstock and H₂Oare typically contacted within a contact zone comprising a vapor phasein which the partial pressure of water is at least about 55 psig.Typically, the partial pressure of water vapor in the contact zone is atleast about 150 psig and, more typically, at least about 175 psig.

2. Pressure Change

As noted, the abrupt change in pressure provided by withdrawing, orremoving pretreated feedstock from a contact zone into a receiving zoneor vessel of reduced pressure degrades thelignin-hemicellulose-cellulose complex. Typically, the pretreatedbiomass feedstock is released from the contact zone to an environment ofatmospheric pressure. Pressure changes associated with such treatmentmay typically be at least about 100, at least about 120, or at leastabout 150 psig. Releasing pretreated feedstock from the contact zone inthis manner may be utilized in suitable pretreatment, but may raise oneor more issues. For example, abrupt pressure changes to atmosphericpressure may release one or more volatile components (e.g., acetic acid,furfural, and hydroxymethyl furfural) and may promote fiber degradation.Optionally, the volatile components may be removed and recovered during,for example, water washing, lignin extraction, hydrolysis, and/orfermentation. Accordingly, in various preferred embodiments, pretreatedfeedstock is removed from the contact zone into a suitable receivingzone or vessel comprising an inlet at which the pressure is aboveatmospheric pressure. More particularly, in various preferredembodiments, to maintain adequate and preferably rapid depressurizationto provide effective degradation of the fiber structure and release ofone or more volatile components, the pressure at the outlet, or exit ofthe steam contact zone and the inlet of the receiving zone typicallydiffers by less than about 100 psig, more typically less than about 75psig and, still more typically, less than about 50 psig.

Generally, as the pressure within the contact zone increases,degradation of feedstock fibers increases upon release of pressure.Degraded fibers may introduce issues in subsequent processing. Forexample, degraded fibers may be more difficult to filter aftersubsequent washing. Thus, in various preferred embodiments, the pressurewithin the contact zone is controlled to preferably avoid substantialdegradation of the feedstock fibers.

At a given partial pressure of water vapor, total pressure in thecontact zone may increase based on the presence of non-condensablecomponents of the feedstock (e.g., air). Thus, in various preferredembodiments, as shown in FIG. 1, the pressure within the contact zonemay be controlled by release of at least a portion of a vapor phasepresent within the contact zone of steam treatment vessel 37 in the formof a flash steam 41. Generally, the mass ratio of flash stream removedfrom the pretreatment vessel to the total proportion of steam introducedinto the vessel is at least about 0.1:1, at least about 0.15:1, or atleast about 0.20:1. Typically, the mass ratio of flash steam 41 removedfrom the vessel to the steam introduced thereto is from about 0.15:1 toabout 0.5:1 and, more typically, from about 0.2:1 to about 0.4:1.

3. Temperature

The temperature of steam introduced into the vessel and/or thetemperature within the vessel and/or contact zone is typically fromabout 160° C. to about 220° C., more typically from about 170° C. toabout 210° C. and, still more typically, from about 180° C. to about200° C.

In various preferred embodiments, distribution of the moisture (e.g.,water vapor of steam treatment) throughout the acid-impregnatedfeedstock is generally uniform and, more preferably, substantiallyuniform. Uniform moisture distribution is currently believed to promoterelatively uniform temperature throughout the contact zone andrelatively uniform temperature of the feedstock. Thus, typically thefeedstock is brought to a target temperature within the contact zone bydistribution of steam throughout the feedstock such that the averagetemperature of a significant portion of the feedstock does not vary froma target temperature to any significant degree. For example, in variouspreferred embodiments, the average temperature of a region of thebiomass feedstock (e.g., a portion of the feedstock constituting atleast about 5% by weight, at least about 25% by weight, or at leastabout 50% by weight of the feedstock) does not differ by more than 5° C.from the target temperature. By way of further example, the averagetemperature of a region of the biomass constituting at least about 60%by weight, or at least about 75% by weight of the feedstock does notdiffer by more than 5° C. or no more than 3° C. from the targettemperature. To promote even temperature distribution throughout thevessel and/or contact zone, various controls are utilized. For example,preferably the total solids content of the feedstock introduced into thevessel and/or contact zone is maintained at from about 30 wt % to about70 wt % (e.g., from about 40 wt % to about 60 wt %). Having a feedstockof total solids content within this range promotes even heating of theacid-impregnated feedstock by direct steam injection as higher moisturecontent feedstocks can result in formation of a large amount ofcondensate on the feedstock that hinders steam penetration and heattransfer throughout the feedstock. If necessary, the feedstock may bedewatered by removing excess acidic liquid medium using a mechanicalsolid/liquid separation device such as a dewatering screw press. Plugscrew feeders commonly used in connection with pretreatment digesters(e.g., continuous pretreatment digesters) may be utilized as thedewatering device. In addition, multiple steam nozzles may be utilizedto promote relatively quick injection of steam into the pretreatmentvessel. For example, in connection with batch pretreatment digesterspreferably multiple steam nozzles are placed at lower portions of thedigester and others are placed at the height of the reactor such thatwhen the valves are opened initially there will be direct contactbetween steam and feedstock mass settled in the vessel. It is currentlybelieved that venting of non-condensable gases contributes tomaintaining the vapor temperature near the temperature of the inputsteam, thereby contributing to even temperature distribution throughoutthe biomass feedstock.

The temperature within the pretreatment vessel may also be controlled tocontribute to venting of one or more volatile components generatedduring the abrupt pressure changes of the acid-impregnated feedstock toatmospheric pressure. For example, furfural has been reported to remainvolatile and therefore able to be removed by venting at temperatures inexcess of 110° C. (e.g., greater than about 120° C.). Thus, in variouspreferred embodiments the temperature of the steam treatment vessel ismaintained above this level to promote venting of furfural andnoncondensable gas.

4. Residence Time

As the residence time of the acid-impregnated feedstock in the vesselfor steam treatment increases, degradation of cellulose and/orhemicellulose to undesired products may be observed. For example, in thecase of cellulose, degradation products such as hydroxymethyl furfuralmay be formed during periods of prolonged treatment. Accordingly, theresidence time within the reactor is typically selected to provide anincrease in cellulose bioavailability and/or solubilizing hemicellulosewithout resulting in product degradation.

Generally, the acid-impregnated feedstock and H₂O (i.e., steam) arecontacted for between about 1 and about 60 minutes, more generallybetween about 1 and about 30 minutes and, still more generally, betweenabout 1 and about 20 minutes. Typically, the acid-impregnated feedstockand H₂O are contacted for between about 1 and about 10 minutes, moretypically between about 2 and about 6 minutes and, still more typically,between about 3 and about 5 minutes.

5. Particle Size

In addition to impacting acid impregnation, the size of the particulatesolids may impact the effectiveness of steam pretreatment. For example,as particle size increases, the bulk density of biomass solids to betreated tends to decrease. Accordingly, the payload and/or costeffectiveness of the steam pretreatment operation may suffer. Inaddition, an increase in solids particle size and the concomitantdecrease in exposed solids surface area may negatively impactdistribution of the acid present in the acid-impregnated feedstock, andlikewise hinder distribution of steam throughout the acid impregnatedfeedstock. Additionally or alternatively, it is currently believed thatas the size of particulates that make up the biomass generallydecreases, the void fraction throughout the acid-impregnated biomassfeedstock generally increases, which promotes distribution of the streamthroughout the feedstock particles. Thus, acid-pretreated feedstockcontaining a significant fraction of particles within the above-notedpreferred particle size ranges provides advantages during subsequentsteam treatment. While the void fraction throughout the feedstockparticles generally promotes distribution of steam throughout, it iscurrently believed that an uneven distribution of the void fraction mayprovide relatively large portions of void fraction that effectively trapa portion of the steam introduced into the reactor and that the steamtrapped throughout this void fraction does not contribute to anysignificant degree to pretreatment of the biomass. It is currentlybelieved that uneven distribution of the void fraction may occur inconnection with feedstock comprising a significant fraction ofrelatively large particles or a significant fraction of relatively smallparticles. Accordingly, acid-pretreated feedstock likewise preferablycontains a significant fraction of particles within the above-notedpreferred particle size ranges to preferably avoid an excessively unevendistribution of void fraction.

In addition, steam treatment of the acid-impregnated feedstock typicallyreduces the size of the particulate solids of the acid-impregnatedfeedstock. During subsequent hydrolysis (e.g., enzymatic hydrolysis ofcellulose using a cellulase enzyme), particulate solids of reduced sizemay provide an increase in exposed surface area of cellulose and/orhemicellulose that would otherwise be provided by the particulate solidsof the feedstock prior to steam treatment.

6. Equipment

The form of the vessel, or reactor utilized for steam treatment is notnarrowly critical and may be selected by one skilled in the artdepending on the intended process conditions. Steam treatment may bepracticed on a batch or continuous basis. For example, in the case ofbatch operations, the vessel may be in the form of a stirred ornon-stirred tank reactor. In the case of continuous steam treatmentoperations, the vessel may be in the form of a continuous horizontalscrew-fed mixer or a vertical vessel. Continuous operation may provideone or more advantages including, for example, avoiding the need tode-pressurize and re-pressurize the contact zone between batches, whichresult in the requirement of larger reactor volumes. Generally,acid-impregnated feedstock is introduced into the steam treatment vesselusing conventional apparatus known in the art including, for example, afeeder such as a plug screw feeder.

7. Two-Stage Pretreatment

Further in accordance with the present invention and, more particularly,the process depicted in FIG. 1, the conditions of elevated temperatureand pressure to which the acid-impregnated feedstock is subjected may becontrolled to promote advantageous dispersion of acid throughout thefeedstock. More particularly, the elevated temperature and pressureconditions may comprise a plurality of intervals, or stages of varyingconditions and, in various preferred embodiments, include two stages ofdiffering temperature and pressure. For example, in various preferredembodiments, acid-impregnated feedstock and steam (i.e., H₂O) arecontacted within a suitable vessel as described above under a first setof conditions (i.e., an “initial steaming period”) for purposes ofbreak-down of the cellulose-hemicellulose-lignin complex and hydrolysisof xylan. This first set of conditions is generally within the range ofpretreatment conditions provided elsewhere herein and typically providesrapid heating of the acid-impregnated feedstock. More particularly, inaccordance with various preferred embodiments, the temperature of thesteam treatment vessel is typically maintained at from about 150° C. toabout 240° C. and, more typically, at from about 160° C. to about 230°C. Typically, the acid-impregnated feedstock is subjected to a saturatedsteam pressure of from about 55 to about 470 psig and, more typically,from about 75 to about 380 psig during this initial period. To provide avessel or contact zone under these conditions, typically steam under apressure of at least about 75 psig, or at least about 100 psig isintroduced into the vessel while air is purged from the vessel by, forexample, venting of the vessel. After purging of air from the vessel iscomplete, any outlets of the vessel are closed and introduction of thepressurized steam into the vessel continues to achieve the desiredconditions. In various embodiments (e.g., when the pretreated feedstockis subjected to relatively low) the contact time of acid-impregnatedfeedstock and steam under the first set of conditions includingadditional steam introduction and purging of the vessel is typicallyfrom about 1 to about 45 minutes and, more typically, from about 1 toabout 30 minutes.

For batch pretreatment, once the first stage of steam treatment iscompleted, introduction of pressurized steam to the vessel isdiscontinued and the pressure within the vessel, or contact zone isreduced, and the second-stage of pretreatment is conducted at reducedpressure and temperature before the contents of the pretreatment vesselare explosively discharged into a collection vessel upon completion ofthe second stage of pretreatment. For continuous pretreatment, once thefirst stage of steam pretreatment is completed, the partially pretreatedfeedstock is transferred into a second-stage vessel in which thepartially pretreated feedstock is subjected to reducedpressure/temperature conditions. Regardless of whether pretreatment isconducted as a batch or continuous process, upon completion of thesecond-stage of pretreatment, the pretreated feedstock is explosivelydischarged into a collection vessel. Generally, the pressure within thecollection vessel is maintained at slightly above atmospheric pressureto prevent ingestion of contaminants from outside the vessel underambient pressure by opening one or more outlets, or vents of the vessel,or by quenching the flash steam with water spray, or by a combination ofventing and water spray. Typically, during the second stage ofpretreatment conditions, the pressure within the vessel, or contact zonerepresents a reduction in pressure of at least about 30 psig and, moretypically, at least about 50 psig (e.g., about 75 psig or greater) ascompared to the pressure within the vessel or contact zone during thefirst stage. For example, preferably the saturated steam pressure withinthe vessel or contact zone during the second stage is from about 25 psigto about 150 psig and, more preferably, from about 50 psig to about 100psig. Additionally or alternatively, the temperature within the vesselor contact zone of the second stage is preferably from about 130 toabout 185° C. and, more preferably, from about 145 to about 170° C. Thepurpose of the second stage (i.e., venting the steam or cooling thepretreated feedstock by adding lower temperature water or other liquidsor solutions) is further hydrolysis of xylan. This stage is typicallyconducted over a period of from about 0.1 to about 5 minutes and, moretypically, conducted over a period of from about 0.3 to about 3 minutes.Once the second stage is complete, pretreated feedstock is expelled fromthe vessel (i.e., as described above and in connection with the processdepicted in FIG. 1.).

Regardless of batch or continuous operation, the primary purpose of thesecond lower temperature and pressure stage of pretreatment ishydrolysis of oligomeric sugars generated during the first stage ofpretreatment. During batch two-stage pretreatment, the period of ventingor pressure reduction of the vessel releases volatiles such as furfural,hydroxymethyl furfural and acetic acid.

Two-stage pretreatment in accordance with the present invention may alsobe conducted by the following method. In the first stage,acid-impregnated feedstock is subjected to conditions within a contactzone effective for solubilizing hemicellulose and producing a streamtreated feedstock. In particular, the conditions within the first stageare effective for providing a liquid fraction within the first stage, orcontact zone comprising xylose. Generally, acid-impregnated feedstock iscontacted with saturated steam at pressures ranging from about 75 psigto about 250 psig or from about 100 psig to about 200 psig. Typically inthe first stage, the acid-impregnated feedstock is contacted withsaturated steam at a pressure of from about 140 psig to about 170 psig.The temperatures to which the acid-impregnated feedstock are subjectedin the first stage of pretreatment are generally from about 140° C. toabout 230° C. or from about 160° C. to about 200° C. Generally, thefirst stage of elevated temperature/pressure conditions is conducted forfrom about 1 to about 15 minutes, and typically for from about 2 toabout 10 minutes.

During the first stage of pretreatment, non-condensable vapor components(e.g., air entrained in the acid-impregnated feedstock) and volatilecomponents generated during the steam treatment (e.g., acetic acid andfurfural) may be continuously removed from the vessel by venting of thevessel combined with introduction of fresh steam to maintain thepressure within the vessel substantially constant. Generally, the ventnozzles are located to provide venting of vapor components withoutventing of feedstock fibers.

Upon completion of the first stage of pretreatment, the steam treatedfeedstock is then subjected to further conditions in a second zoneeffective for additional solubilizing of hemicellulose, hydrolyzing ofoligosaccharides, and producing a volatilized fraction of the steamtreated feedstock. The pressures to which the steam treated feedstock issubjected in this second zone are lower than the pressure within thecontact zone of the first stage. For example, typically the steamtreated feedstock is subjected to pressures of from about 5 to about 50psig, from about 5 psig to about 40 psig, or from about 10 to about 15psig during this second stage of pretreatment. In this manner, thesecond stage of pretreatment may be described as conducted in adepressurization zone. The pressures during the second stage and withinthe depressurization zone generally correspond to temperatures of fromabout 110° C. to about 150° C., more typically from about 110° C. toabout 140° C. (e.g., from about 110° C. to about 120° C.).

In accordance with the two-stage method of pretreatment, a volatilizedfraction of the steam treated feedstock is removed from thedepressurization zone. The volatilized fraction generally comprisesfurfural, acetic acid, steam, or a combination thereof. Releasing thevolatilized fraction controls the pressure and temperature within thedepressurization zone. Release of the volatilized fraction andpressure/temperature control may be conducted using a pressure controlvalve or damper on the exhaust gas line.

The conditions within the depressurization zone are effective forsolubilizing hemicellulose and, thus, provide a liquid fraction withinthe depressurization zone containing xylose. The conditions within thedepressurization zone are controlled to provide continued solubilizationof hemicellulose, but without excessive degradation of cellulose andsugars. Typically, the conditions of the depressurization zone arecontrolled to provide a xylose content of the liquid fraction thatrepresents a xylose yield of at least about 60%, at least about 70%, orat least about 80% based on the hemicellulose content of the cellulosicbiomass feedstock. Additionally or alternatively, the conditions of thedepressurization zone provide a xylose content of the liquid fraction inthe depressurization zone that is typically at least 5%, 10%, 20%, or30% higher than the xylose content of the liquid fraction in the contactzone.

Temperature control within the depressurization zone allows for ventingof volatile components such as, for example, steam, acetic acid, andfurfural. Venting of furfural in the vapor phase avoids reaction offurfural in the liquid phase with sugars and/or formation of inhibitorsof enzymatic hydrolysis. Thus, preferably the conditions within thedepressurization zone maintain furfural in the vapor phase. Sincefurfural is known to exist in the vapor phase at temperatures in excessof 110° C., preferably the conditions within the second stage vesselmaintain the temperature above 110° C. to allow for venting of thefurfural volatile component. Although preferably maintained above 110°C. to maintain the furfural component in a volatile state, temperaturesat or within the lower of the above-noted ranges (e.g., from about 110°C. to about 115° C.) are often preferred in order to maximize thetemperature difference between the fibers and feedstock and the heatabsorbing liquid, which may serve to increase the rate at which thefibers are cooled.

Advantageously, the control of pressure and temperature within thedepressurization may be conducted only through releasing of a portion ofthe volatilized fraction and preferably is conducted only throughreleasing a portion of the volatilized fraction. That is, the two-stagepretreatment method of the present invention does not utilize anyadditional liquid media for temperature control and/or pH adjustment.

This two-stage pretreatment is preferably conducted in a continuousmanner. That is, preferably the acid impregnated feedstock iscontinuously subjected to the above-noted conditions within a contactzone for the first stage of pretreatment while steam treated feedstockprovided by the first stage of pretreatment is continuously subjected tothe above-noted conditions within the depressurization zone and avolatilized fraction is released from the depressurization zone. Thetwo-stage process may utilize a single or multiple vessels. That is, invarious embodiments the contact zone for the first stage of pretreatmentand the depressurization zone for the second stage of pretreatment arecontained in a single vessel. In various other embodiments, the contactzone and depressurization zone are contained in separate vessels. Forexample, in various embodiments the first stage is conducted in avertical or horizontal pretreatment digester and the second stage isconducted in a suitable vessel such as, for example, a blow tank.

Upon completion of the second stage of pretreatment, the feedstock iscontinuously discharged from the vessel (e.g., utilizing a screwconveyor feeding a blow valve) and introduced into the vessel for thesecond stage of pretreatment. The second step of steam treatment isconducted in a suitable vessel such as, for example, a blow tank.

Utilizing a high pressure blow tank is currently believed to provideadditional hydrolysis of xylan to xylose and dissipation of heat fromthe fibers. In addition, generally there is a period of time afterdischarge of pretreated feedstock from the reactor before thetemperatures of the solid and liquid fractions reach equilibrium.Typically, the solids fraction is cooled as heat is transferred byconvection and/or conduction to the liquid fraction. Thus, as the solidsfraction is cooled, the liquid fraction is heated. During high pressuredischarge, a significant fraction, and preferably substantially all theheat is released from the liquid fraction by evaporation and thereforeas heat is transferred from the solids to liquid fraction the heatrecovered by the liquid fraction is release through this evaporation.After a suitable period to time, the pretreated material is thensubjected to further treatment including, for example, conditioning asdetailed elsewhere herein.

C. Pretreated Feedstock

Again with reference to FIG. 1, pretreated feedstock 45 is in the formof a mixture comprising feedstock fibers and including a solids fractionand a liquid fraction. Typically, the pretreated feedstock is in theform of a slurry comprising insoluble fibers, water, and solublematerials and having a moisture content of from about 40 wt % to about80 wt %, more typically from about 50 wt % to about 70 wt %. Thetemperature of the pretreated feedstock exiting the steam treatment zoneis not narrowly critical, but is typically from about 80° C. to about120° C. and, more typically, from about 90° C. to about 110° C. The pHof the pretreated feedstock is typically less than about 4, less thanabout 3.5, or less than about 3 (e.g., from about 1 to about 2.5).

1. Solids Fraction

The water-insoluble solids fraction of the pretreated feedstockgenerally comprises those solids of the acid-impregnated feedstock thatare not solubilized during acid and steam treatment. The solids fractionof the pretreated feedstock generally comprises cellulose, unsolubilizedlignin, unsolubilized hemicellulose, and unsolubilized ash, andgenerally constitutes at least about 30 wt %, at least about 40 wt %, orat least about 50 wt % of the pretreated feedstock. For example,typically the water-insoluble solids fraction constitutes from about 40wt % to about 80 wt % of the pretreated feedstock, more typically fromabout 50 wt % to about 75 wt % and, still more typically, from about 60wt % to about 75 wt % of the pretreated feedstock. The composition ofthe solids fraction of the pretreated feedstock generally corresponds tothe composition of the acid-impregnated feedstock, adjusted forbreak-down of the cellulose-hemicellulose-lignin complex. For example,in various embodiments at least about 10%, at least about 20%, at leastabout 30%, or at least about 40% of the polysaccharide content of thepretreated feedstock is solubilized and can be found in the liquidfraction of the pretreated feedstock.

Generally, cellulose constitutes at least about 30 wt %, at least about40 wt %, or at least about 50 wt % of the water-insoluble solidsfraction. Typically, cellulose constitutes from about 35 wt % to about65 wt %, more typically from about 40 wt % to about 60 wt % and, stillmore typically, from about 45 wt % to about 55 wt % of the solidsfraction. Cellulose contents of the solids of the pretreated feedstockmay be determined by conventional means known to one skilled in the artincluding, for example, concentrated acid hydrolysis of cellulose toglucose and determining the glucose released by High Performance LiquidChromatography (HPLC).

One indicator of effective pretreatment (i.e., effective break down ofthe cellulose-hemicellulose-lignin complex to provide celluloseavailable for preparation of fermentable sugars) is a solids fractionthat includes a significant fraction of the initial cellulose content ofthe feedstock. Accordingly, additionally or alternatively, in variouspreferred embodiments the pretreated feedstock solids fraction typicallycomprises at least about 40 wt %, more typically at least about 45 wt %and, still more typically, at least about 50 wt % of the initialcellulose content of the feedstock. As detailed in the working examplesand mass balances provided herein, such recoveries of cellulose inpretreated feedstocks may be provided by a variety of combinations ofpretreatment parameters.

The water-insoluble solids fraction also typically comprises lignin. Forexample, typically lignin constitutes at least about 20 wt %, at leastabout 25 wt %, or at least about 30 wt % of the water-insoluble solidsfraction. Additionally or alternatively, the water-insoluble solidsfraction of the pretreated feedstock typically comprises up to about 75wt % or up to about 95 wt % of the initial lignin content of thefeedstock.

As detailed below, pretreatment generally solubilizes a significantfraction of hemicellulose, but a fraction of hemicellulose may bepresent in the water-insoluble solids fraction of the pretreatedfeedstock. For example, hemicellulose may constitute up to about 4 wt %,up to about 6 wt %, or up to about 8 wt % of the water-insoluble solidsfraction. More particularly, up to about 6 wt %, up to about 10 wt %, upto about 20 wt %, or up to about 25 wt % of the initial hemicellulosecontent of the feedstock may be present in the water-insoluble solidsfraction of the pretreated feedstock.

2. Liquid Fraction

The liquid fraction of the pretreated feedstock typically comprisessolubilized hemicellulose, solubilized cellulose, and solubilizedcomponents provided by degradation of lignin. Pretreatment preferablyincreases the bioavailability of the feedstock which may be indicatedby, for example, degradation of the cellulose-hemicellulose-lignincomplex and/or break down of cellulose and/or hemicellulose intofermentable sugars. For example, in accordance with the process depictedin FIG. 1, lignin and/or various soluble fermentable sugars aretypically present in the liquid fraction of the pretreated feedstock.More particularly, fermentable sugars (e.g., glucose, xylose, arabinose,mannose, galactose, and various oligomers thereof) generally constituteat least about 30 wt %, at least about 50 wt %, or at least about 75 wt% of the water-soluble fraction of pretreated feedstock. Typically,fermentable sugars constitute from about 50 to about 95 wt % and, moretypically, from about 60 to about 90 wt % of the water-soluble fractionof the pretreated feedstock. Additionally or alternatively, preferablyfermentable sugars (e.g., xylose) solubilized in the liquor portion ofthe pretreated feedstock represent a yield (basis fermentable sugarcontent of the feedstock) of at least about 70%, at least about 80%, orat least about 90%.

Lignin typically constitutes at least about 0.5 wt %, more typically atleast about 1 wt % and, still more typically, at least about 4 wt % ofthe water-soluble fraction of the pretreated feedstock. Additionally oralternatively, as noted, the liquid fraction may also comprise solublelignin-derived components. For example, the liquid fraction may comprisewater-soluble lignin-derived phenolic components and relatively lowmolecular weight lignin degradation products.

III. Conditioning

Again with reference to FIG. 1, pretreated feedstock 45 is introducedinto conditioning vessel 49 along with conditioning stream 53.Pretreated feedstock may comprise one or more components that willinhibit hydrolysis of hemicellulose and/or cellulose. These componentsmay also inhibit fermentation of sugars derived from hemicelluloseand/or cellulose. For example, lignin is often broken down intowater-soluble phenolic compounds during pretreatment. Pretreatedfeedstock may also comprise degradation products of hemicellulose and/orcellulose hydrolysis. For example, during pretreatment hemicelluloseand/or cellulose may be hydrolyzed to form a sugar that may be degradedto form one or more of hydroxymethyl furfural (HMF), furfural, and/oracetic acid. In accordance with the present invention, advantageouslyconditioning for inhibitor removal is conducted prior to enzymatichydrolysis for the primary purpose of hydrolysis of either hemicelluloseor cellulose to provide fermentable sugars. It is currently believedthat conditioning in this manner contributes to maximum fermentablesugar and ethanol yields on both hemicellulose and cellulose.

Generally, the pretreated feedstock is contacted with material suitablefor absorbing and/or forming a complex with one or more of theinhibitors and/or neutralization of one or more inhibitors. In variousembodiments in which the feedstock comprises water-soluble phenoliccompounds, the process comprises contact of the feedstock with materialthat adsorbs and/or forms a complex with one or more phenolic compounds.For example, the pretreated feedstock or its liquor portion may becontacted with an alkali metal hydroxide or oxide that forms a phenatesalt. Suitable alkali sources include sodium hydroxide, calciumhydroxide, ammonium hydroxide, calcium oxide (lime), and combinationsthereof The phenate salts thus formed may be removed from the pretreatedfeedstock in accordance with means known in the art including, forexample, filtration. By way of further example, the feedstock may becontacted with protein-containing material that will absorb the phenoliccompounds and/or form a complex and/or adduct with the phenoliccompounds. Various protein-containing materials (e.g., enzymes, yeastcells and fermentation broths generated during enzyme production) aresuitable for this purpose. Enzymes (e.g., lacase) may providedegradation of phenolic compounds. In addition, protein-containingmaterial derived at other process stages may be utilized. For example,thin stillage and cereal mash produced as described elsewhere herein maybe used for this purpose. Metal salts and/or protein-containingmaterials may also be used in treatment for the purpose of complexingand/or absorbing hemicellulose and/or cellulose degradation products.Suitable metal salts (e.g., ferrous sulfate and magnesium sulfate) maybe introduced into the liquor fraction of the pretreated feedstock at aconcentration of from about 0.05 to about 1 millimole/L (mmol/L).

Typically, pretreated feedstock is conditioned without any intermediatesteps between steam treatment and addition of conditioning agents.However, since further degradation products may form in the pretreatedfeedstock at elevated temperature, the temperature of the feedstockprior to conditioning is preferably maintained at no more than about140° C., or no more than about 120° C. If necessary, the pretreatedfeedstock may be cooled prior to conditioning to bring its temperaturewithin these ranges.

Conditioning stream 53 is typically in the form of an aqueous liquidmedium comprising one or more of the above-noted components. Typically,one or more components are present in the stream at a proportion of fromabout 0.25 to about 2.5 wt % and, more typically, at a proportion offrom about 0.5 to about 1 wt %. Generally, the mass ratio of stream 53to pretreated feedstock 45 introduced into conditioning vessel 49 is atleast about 0.05:1, or at least about 0.1:1. For example, typically themass ratio of stream 53 to pretreated feedstock 45 introduced into theconditioning vessel is from about 0.05:1 to about 0.25:1 and, moretypically from about 0.1:1 to about 0.2:1. Contact of the pretreatedfeedstock with the conditioning stream within the conditioning vesselforms a conditioned feedstock 57. With respect to the principalcomponents of value, i.e., cellulose, hemicellulose, and sugars, thecomposition of the conditioned feedstock generally corresponds to thatof the pretreated feedstock, with the proportions of the componentsreduced based on dilution of the pretreated feedstock by mixing with theconditioning stream within the conditioning vessel. It is currentlybelieved that conditioning has little, if any, impact on, for example,the cellulose, hemicellulose, solubilized sugar and/or lignincomposition of the pretreated feedstock. Lignin degradation products maybe removed from the pretreated feedstock by virtue of complexing orreaction with a component of the conditioning stream. For example,generally the conditioned feedstock 57 is in the form of a slurrycomprising a solids fraction and a liquid fraction, and having a totalsolids content of at least about 10 wt %, at least about 20 wt %, or atleast about 30 wt %. For example, typically the solids content of theconditioned feedstock is from about 10 wt % to about 50 wt % and, stillmore typically, from about 20 wt % to about 40 wt %.

IV. Enzymatic Hydrolysis

Again with reference to FIG. 1, conditioned feedstock 57 is introducedinto vessel 61 and contacted with an enzyme-containing stream 65 forenzymatic hydrolysis to yield glucose and hemicellulose-derivedfermentable sugars. Suitable enzymes include various hemicellulase andcellulase enzymes generally produced by fermenting a microorganism ofthe Trichoderma genus, including, for example, xylanase, β-xylosidase,acetyl esterase, and α-glucuronidase, endo- and exo-glucannase,cellobiase, and combinations thereof. These enzymes may be isolated fromenzyme solutions by fractionation techniques known in the art including,for example, ammonium sulfate precipitation and ultrafiltration, orrecovered from whole enzyme production broth.

Hemicellulose is primarily composed of polysaccharides comprising fiveand six carbon sugars including, for example, glucose, xylose, mannose,galactose, rhamnose, and arabinose. The hemicellulose portion oflignocellulosic biomass is typically primarily composed of xylose (amonosaccharide containing five carbon atoms and including an aldehydefunctional group). Accordingly, the pretreated feedstock is typicallycontacted with a xylanase enzyme (enzyme stream 65 in FIG. 1). Xylanasesare a class of enzymes that degrade the linear polysaccharideβ-1,4-xylan into xylose. Hemicellulose also typically comprisesarabinose, also a monosaccharide containing five carbon atoms andincluding an aldehyde functional group.

Enzyme stream 65 generally comprises an enzyme dispersed throughoutand/or dissolved in a suitable liquid medium (e.g., water). Typically,the enzyme stream is in the form of a slurry having a solids content offrom about 1 to about 20 wt % and, still more typically, a solidscontent of from about 5 to about 15 wt %. The mass ratio of enzymestream to conditioned feedstock is generally from about 0.005:1 to about0.1:1, typically from about 0.005:1 to 0.1:1 and, still more typically,from about 0.007:1 to about 0.05:1.

The configuration of the vessel for contact of the enzyme and pretreatedfeedstock is not narrowly critical and may be readily selected by oneskilled in the art. For example, in various embodiments, the hydrolysisis conducted continuously utilizing a plug flow reactor. Enzymatichydrolysis of hemicellulose may also be conducted as a batch processutilizing a stirred tank reactor. Regardless of the precise nature ofthe process (e.g., batch or continuous), preferably the mixture ofpretreated feedstock and enzyme is agitated to promote contact and,therefore, promote hydrolysis of hemicellulose to simple sugars that maybe fermented to produce ethanol.

The precise conditions of hydrolysis are not narrowly critical, butgenerally are selected and controlled to provide suitable sugar yields.Typically, the enzyme loading in the contact zone is at least about 1Filter Paper Unit (FPU) (i.e., International Units of filter paperactivity in micromoles of glucose per minute) per g glucan or cellulose,more typically at least about 2 FPU per g glucan or cellulose and, stillmore typically, at least about 5 FPU of enzyme per g glucan orcellulose. In various preferred embodiments, the enzyme loading withinthe reactor is from about 2 to about 40 FPU per g glucan or cellulose,from about 4 to about 20 FPU per g glucan or cellulose, or from about 5to about 15 FPU per g glucan or cellulose. The temperature at which thehydrolysis reaction is conducted is not narrowly critical, but typicallyis from about 30° C. to about 70° C. (e.g., about 50° C.). Additionallyor alternatively, the hydrolysis is typically conducted at a pH of fromabout 4 to about 6 and, more typically, from about 4.5 to about 5.5.

Contact of the pretreated feedstock and the hemicellulase enzymegenerally provides a pretreated hydrolyzate 69 comprising a liquid phasecomprising solubilized hemicellulose-derived fermentable sugars and asolid phase comprising cellulose and lignin. For example, typically thesolubilized hemicellulose constitutes from about 10 wt % to about 80 wt% oligomeric sugars. Enzymatic hydrolysis of hemicellulose typically haslittle effect, if any, on the cellulose and lignin portions of thesolids fraction of the pretreated feedstock. Typically, the pretreatedhydrolyzate contains solubilized hemicellulose at a concentration of atleast about 8 wt %, preferably at least about 10 wt % and, morepreferably, at least about 12 wt %. Preferably, enzymatic hydrolysisprovides a hemicellulose-derived sugar yield (e.g., xylose) of at leastabout 70%, more preferably at least about 80% and, still morepreferably, at least about 90% (basis hemicellulose content of thepretreated feedstock).

V. Sugar Recovery

Hemicellulose-derived fermentable sugars may be fermented to produceethanol. As shown in FIG. 1, an aqueous fraction comprising one or morehemicellulose-derived sugars (e.g., C₅ sugar(s)) is removed, orseparated from the pretreated hydrolyzate 69 to provide fermentablesugars that may be utilized for fermentation to ethanol as detailedelsewhere herein. For removal of the C₅ sugar fraction, pretreatedhydrolyzate 69 is introduced into sugar recovery vessel or device 73which comprises a solids/liquid separation instrumentality such as,e.g., a screen, filter, centrifuge, settler, percolator, extractioncolumn, flotation vessel, or combination thereof. The aqueous fractioncomprising hemicellulose-derived fermentable sugars is combined with aliquid medium (e.g., water) 77 to form a hemicellulose-derived sugarfraction 81. The precise composition of the liquid medium is notnarrowly critical. However, in various preferred embodiments, thewashing liquid is supplied by recycle from elsewhere in the process. Forexample, the liquid medium (e.g., water) may be provided by thinstillage produced as detailed elsewhere herein. In various preferredembodiments, washing for sugar recovery includes counter-current contactof the washing liquid and aqueous fraction in a suitable apparatus.

Hemicellulose-derived sugar fraction 81 is typically in the form of aslurry, or filtered liquor having a dissolved solids content of at leastabout 5 wt %, or at least about 6 wt %. Typically, the solids content ofthe hemicellulose-derived sugar fraction is from about 5 to about 10 wt% and, more typically, from about 7 to about 9 wt %. The total sugarcontent (e.g., glucose, xylose, arabinose, mannose, and galactose) ofthe hemicellulose-derived sugar-rich fraction 81 is generally at leastabout 5 wt %, or at least about 6 wt % (basis total fraction weight).Typically, the sugar content of the hemicellulose-derived sugar-richfraction is from about 5 to about 10 wt % and, more typically, fromabout 6 to about 9 wt %. Generally, the xylose content of thehemicellulose-derived sugar-rich fraction is at least about 2.5 wt %, orat least about 4 wt % (basis total fraction weight). Typically, thexylose content of the hemicellulose-derived sugar-rich fraction is fromabout 2.5 to about 9 wt % and, still more typically, from about 5 toabout 7 wt % (basis total fraction weight).Recovery of thehemicellulose-derived sugar fraction 81 from the pretreated hydrolyzate69 generally provides a residual thickened fraction 85 that typicallycomprises a cake, or slurry comprising a solid phase comprisingcellulose and lignin, and a residual liquid phase comprisinghemicellulose-derived fermentable sugars. Preferably, the solids/liquidseparation is conducted in accordance with conventional methods known inthe art utilizing, for example, a screen, filter, centrifuge, settler,vacuum belt washer, pressure filter, membrane filter, extraction column,flotation vessel, counter-current screw extractor, or screw press.Preferably, sugars are recovered from the pretreated hydrolyzateutilizing a vacuum belt filter or countercurrent screw extractor orextraction column. Additionally or alternatively, recovery of ahemicellulose-derived sugar fraction from the pretreated hydrolyzate maycomprise contacting the hydrolyzate with a suitable extraction medium.

VI. C₅ Sugar Fermentation

In accordance with the present invention and, more particularly, inaccordance with the process depicted in FIG. 1, hemicellulose-derivedfermentable sugars (i.e., C₅ sugars) may be fermented to produceethanol. In particular, these sugars may be converted to ethanol inparallel with fermentation of cellulose-derived sugars (as detailedelsewhere herein). In this manner, the process depicted in FIG. 1provides improved ethanol yield as compared to processes that relysolely on cellulose-derived sugars for ethanol production.

Again with reference to FIG. 1, a portion of the hemicellulose-derived(i.e., C₅) sugar fraction 81 is introduced into a yeast adaptationvessel 89 for production of yeast for fermentation of C₅ sugars.Typically, the portion of the sugar fraction 81 introduced into yeastadaptation vessel 89 constitutes from about 0.5 to about 10 wt % and,more typically, from about 2 to about 6 wt % of the entire C₅ sugarfraction present in enzyme treatment vessel 73.

Along with a portion of the C₅ sugar fraction, yeast culture 93 isintroduced into yeast adaptation vessel 89 to grow yeast forfermentation of the C₅ sugars and/or transform and adapt the yeast to beeffective in fermentation of C₅ sugars.

Suitable yeast include those generally known in the art. In variouspreferred embodiments, the yeast is Pichia stipitis, but various otherspecies of yeast may be utilized. The method of yeast adaptation is notnarrowly critical and generally proceeds in accordance with conventionalmethods known in the art including, for example, as described in Kelleret al. “Yeast Adaptation on Softwood Prehydrolysate,” AppliedBiochemistry and Biotechnology, 1998, Pages 137-148, Volume 70-72, whichthe entire contents of is incorporated herein by reference for allrelevant purposes. Generally, the mass ratio of yeast and C₅ sugarfraction introduced into the yeast adaptation vessel is at least about0.05:1, or at least about 0.1:1. For example, typically the mass ratioof yeast and C₅ sugar fraction is from about 0.05:1 to about 0.25:1 and,more typically, from about 0.1: to about 0.2:1. Yeast 93 is typically inthe form of a solution or slurry of yeast dissolved in or dispersedthroughout a suitable liquid medium. For example, in various embodimentsyeast 93 has a total solids content of from about 1 to about 20 wt %, orfrom about 5 wt % to about 15 wt %. Typically, the yeast-containingliquid medium contains the yeast at a concentration of from about 0.60to about 150 g/L, or from about 0.80 to about 120 g/L.

Although the foregoing and following discussion focuses on use of yeastin fermentation of C₅ sugars, it is to be understood that any organism(e.g., yeast or bacteria) suitable for metabolizing C₅ sugars may beutilized in the process of the invention.

Combining yeast and the C₅ sugar fraction provides a yeast inoculum 97for use in fermentation of C₅ sugars. Yeast inoculum is generally in theform of a slurry comprising the yeast recovered from yeast adaptationvessel. More particularly, yeast inoculum is typically in the form of aslurry of yeast dissolved in and/or dispersed throughout a liquidmedium. Typically, yeast inoculum has a yeast concentration of fromabout 15 g/L to about 25 g/L and, more typically, a yeast concentrationof from about 17 g/L to about 22 g/L. Additionally or alternatively,typically the yeast inoculum slurry a total solids content of from about1 wt % to about 10 wt % and, more typically, a total solids content offrom about 2 wt % to about 6 wt %.

As shown in FIG. 1, along with yeast 93, supplement 91 is introducedinto yeast adaptation vessel 89. The supplement is generally in the formof a solution and comprises syrup, cane molasses, beet molasses, water,urea, commercial yeast nutrients, or a combination thereof. Althoughshown in FIG. 1, it is to be understood that use of supplement in yeastadaptation vessel is not required in accordance with the presentinvention. Also, in various preferred embodiments, to promote yeast cellgrowth and adaptation to inhibitors, filtered air is supplied to theadaptation vessel (not shown in FIG. 1) to provide advantageous oxygentransfer required for yeast growth.

Yeast inoculum 97 is introduced into fermentation vessel 101 along withC₅ sugar fraction 81 recovered from the enzyme treatment vessel but notintroduced into the yeast adaptation vessel. The relative proportions ofyeast inoculum and C₅ sugar fraction introduced into the fermentationvessel are not narrowly critical and depend on a variety of factorsincluding, for example, the composition of each stream. For example, asthe proportion of yeast in the inoculum increases and/or the proportionof C₅ sugars in the sugar fraction increases, reduced proportions ofinoculum may be required to obtain suitable yields of ethanol on C₅sugars. Typically, however, the mass ratio of yeast solids to C₅ sugarfraction is from about 0.01:1 to about 1:1 and, more typically, fromabout 0.05:1 to about 0.5:1 (e.g., from about 0.05:1 to about 0.1:1).Typically, the concentration of yeast in the yeast inoculum is fromabout 0.15 to about 30 g/L and, more typically, from about 15 to about25 g/L.

The configuration of the fermentation vessel is not narrowly criticaland may be readily selected from conventional apparatus by one skilledin the art. The conditions of the contact of the C₅ sugar fraction withthe yeast inoculum are likewise not narrowly critical. Typically,however, the C₅ sugar fraction and yeast inoculum are contacted at atemperature of from about 20° C. to about 60° C. and, more typically, ata temperature of from about 25° C. to about 40° C.

Again with reference to FIG. 1, contacting the C₅ sugar fraction andyeast inoculum forms a C₅ fermentate 101. Generally, C₅ fermentate is anaqueous mixture of water, ethanol, and unconverted sugars of the C₅sugar fraction. Typically, the concentration of ethanol in the C₅fermentate is at least about 1 wt %, at least about 2 wt %, or at leastabout 4 wt %. However, the composition of the C₅ fermentate generallyvaries depending on, for example, the composition of the sugar fractionintroduced into the fermentation vessel and the relative proportions ofyeast inoculum and C₅ sugar fraction introduced into the vessel.Preferably, the composition of the C₅ fermentate represents suitableethanol yields based on the fermentable sugar content of the C₅ sugarfraction. For example, generally the ethanol yield of the C₅ fermentateis at least about 50%, at least about 60%, or at least about 70%. It iscurrently believed that ethanol yields satisfying these limits, andhigher, are achieved in accordance with the process depicted in FIG. 1.The residual sugar content of the C₅ fermentate depends on thecomposition of the C₅ sugar fraction and the ethanol yields achieved,but preferably constitutes no more than about 40 wt % and, morepreferably, no more than about 30 wt % of the fermentable sugar contentof the C₅ sugar fraction. Unfermented sugars may be converted to biogasin an aerobic digestion step, which may be incorporated into thewastewater treatment system.

VII. Lignin Extraction

As noted, recovery of C₅ sugar fraction 81 from enzymatic hydrolyzate 69yields a residual thickened fraction 85 in the form of a cake orconcentrated slurry comprising solid phase cellulose and lignin (i.e.,cellulose/lignin residual fraction). The solids content of thecellulose/lignin residual fraction is typically from about 15 to about45 wt % and, more typically, from about 25 to about 35 wt %. Thecellulose content of the solids fraction is generally from about 35 toabout 55 wt %, and typically from about 40 to about 50 wt %. The solidsfraction of the cellulose/lignin residual fraction typically comprisesvarious sugars including, for example, polysaccharides such as glucan,xylan, arabinan, mannan, and galactan, monosaccharides such as xylose,arabinose, and combinations thereof For example, in various embodiments,the total glucan content of the residual fraction is typically fromabout 35 to about 55 wt %, and more typically from about 40 to about 50wt %. The total xylan content of the residual fraction is typically fromabout 1 to about 7 wt %, and more typically from about 1 to about 3 wt%. Additionally or alternatively, the arabinan content of the residualfraction is typically less than about 1.5 wt % and, more typically, lessthan about 1 wt %. The residual fraction also typically comprisesvarious other fermentable sugars (e.g., mannose and galactose) in aproportion of less than about 1 wt %, and more typically less than about0.5 wt %.

The lignin content of the cellulose/lignin residual fraction isgenerally from about 20 to about 40 wt %, and typically from about 25 toabout 40 wt %. The lignin content of the solids fraction of the residualfraction is typically from about 25 to about 35 wt %, and more typicallyfrom about 30 to about 33 wt %.

As shown in FIG. 1, cellulose/lignin residual fraction 85 is introducedinto lignin extraction vessel or device 105. As noted, effectivepretreatment of the biomass breaks down thecellulose-hemicellulose-lignin complex (e.g., breaks bonds betweenlignin and hemicellulose and/or cellulose). In this manner, and asdetailed elsewhere herein, hemicellulose and cellulose are available forenzymatic hydrolysis to produce fermentable sugars. Similarly,pretreatment provides lignin available for recovery as a further productof the process. Lignin-rich products are suitable for use in a varietyof applications including, for example, as a phenol formaldehyde resinextender in the manufacture of particle board and plywood, manufactureof molding compounds, urethane and epoxy resins, antioxidants, feeds,fuels, pelletizing aids, drilling mud stabilizers, and cement additives.In the process depicted in FIG. 1, lignin is recovered prior toconversion of cellulose to fermentable sugars, and conversion of thecellulose-derived fermentable sugars to ethanol.

The solids fraction of the cellulose/lignin residual fraction 85typically comprises various sugars (e.g., cellulose-derived sugars)including, for example, polysaccharides such as glucan, xylan, andarabinan, monosaccharides such as xylose and arabinose, and combinationsthereof. For example, generally the total sugar content of the solidsfraction is no more than about 60 wt %, no more than about 55 wt %, orno more than about 50 wt %. Typically, the total sugar content of thesolids fraction is from about 40 to about 70 wt % and, more typically,from about 50 to about 65 wt %. More particularly, the total glucancontent of the cellulose/lignin residual fraction is typically fromabout 40 to about 60 wt %, and more typically from about 45 to about 55wt %. The total xylan content is typically from about 1 to about 10 wt%, and more typically from about 1 to about 5 wt % (e.g., from about 1to about 3 wt %). Additionally or alternatively, the arabinan content ofthe cellulose/lignin residual fraction is typically from about 0.5 toabout 3 wt %, and more typically from about 1 to about 2 wt %.

The lignin content of the cellulose/lignin fraction is typically fromabout 25 to about 45 wt %, more typically from about 28 to about 42 wt %and, still more typically, from about 30 to about 40 wt %.

Again with reference to FIG. 1, an extraction solvent 109 is introducedinto lignin extraction vessel or device 105 along with thecellulose/lignin residual fraction 85. The extraction solvent may be inthe form of an organic solvent comprising methanol, ethanol, butanol,acetone, and combinations thereof. The extraction solvent may alsocomprise an alkali metal hydroxide, e.g., sodium hydroxide, potassiumhydroxide, ammonium hydroxide, or a combination thereof In variouspreferred embodiments, the extraction solvent comprises sodium hydroxidedissolved in water and, more particularly, is in the form of an aqueoussolution of sodium hydroxide containing sodium hydroxide at aconcentration of from about 0.5 to about 2 wt %, or from about 0.5 toabout 1 wt %. Neither the conditions of nor the manner of contact of thecellulose/lignin fraction with the extraction solvent are narrowlycritical and are generally conducted in accordance with conventionalmethods known in the art. See, for example, Canadian Patent Nos. 1 267407 and 1 322 366 and U.S. Pat. Nos. 3,817,826; 4,470,851; 4,764,596;4,908,099; and 4,966,650, the entire contents of which are incorporatedherein by reference for all relevant purposes. For example, inaccordance with the embodiment depicted in FIG. 1, the extractionsolvent is an alkaline aqueous medium having a pH of from about 10 toabout 14 (e.g., about 13). Additionally or alternatively, thetemperature of the extraction solvent is typically from about 30° C. toabout 60° C., and more typically from about 40° C. to about 50° C.(e.g., about 45° C.).

Mixing the cellulose/lignin residual fraction and extraction solventwithin an extraction zone of the extraction vessel forms an extractionmixture comprising a liquid fraction comprising lignin (e.g., lignindissolved in the extraction solvent) and a solid phase comprisingcellulose and depleted in lignin relative to the cellulose/ligninresidual fraction. A lignin fraction 113 is separated from theextraction mixture. Lignin typically constitutes at least about 1 wt %,more typically at least about 2 wt % and, still more typically, at leastabout 3 wt % of the lignin fraction. For example, lignin generallyconstitutes from about 1 to about 10 wt %, or from about 2 to about 6 wt% of the lignin fraction. Generally at least about 60 wt %, at leastabout 70 wt %, at least 80 wt %, or at least about 90 wt % of the ligninis soluble in the lignin fraction.

As shown in FIG. 1, lignin extract 113 is introduced into vessel 113Afor recovery of a lignin-rich product from the extract. Recovery of thelignin-rich product from the lignin extract generally proceeds inaccordance with conventional methods known in the art (e.g.,precipitation) as described, for example, in U.S. Pat. No. 4,966,650 toDelong et al., the entire contents of which are incorporated herein byreference for all relevant purposes. As shown in FIG. 1, acid 114 isintroduced into vessel 113A for precipitation of the lignin-rich solidsfrom the lignin extract. In various preferred embodiments, including theembodiment shown in FIG. 1, acid 114 is in the form of a relativelyconcentrated acid. For example, acid 114 may be in the form of asulfuric acid solution containing at least about 50 wt % sulfuric acid,at least about 80 wt % sulfuric acid, or at least about 90 wt % sulfuricacid.

Contacting acid 114 and lignin extract 113 within vessel 113A generallyforms a lignin product mixture 114A comprising lignin precipitates thatis introduced into vessel 115 for removal of moisture from the ligninproduct mixture (e.g., a vessel including a filter and dryer) to form alignin powder product 116 and a waste stream 116A. Removal of moisturefrom the lignin product mixture generally proceeds in accordance withconventional methods known in the art including, for example, by heatingthe mixture to temperatures in excess of about 70° C., or in excess ofabout 90° C.

Lignin product 116 is typically in the form a particulate (e.g., powder)product having a moisture content of no more than about 20 wt %, moretypically no more than about 15 wt %, and preferably no more than about10 wt %. Generally, the lignin content of lignin product 113 is at leastabout 75 wt %, or at least about 80 wt %. Preferably, the lignin contentof the lignin product is at least about 85 wt % and, more preferably, atleast about 90 wt %. One advantage of recovery of a lignin product asshown in FIG. 1 (i.e., prior to recovery and fermentation ofcellulose-derived sugars) is allowing for utilizing less reactors duringenzymatic hydrolysis of cellulose and/or reactors of reduced reactorvolume during enzymatic hydrolysis than typically required inconventional processes.

Again with reference to FIG. 1, a wet cake 117 comprising solid phasecellulose fibers is removed from lignin extraction vessel 105. Thesolids content of the wet cake is typically at least about 10 wt %, moretypically at least about 20 wt % and, still more typically, at leastabout 30 wt %. The solids fraction of the wet cake 117 generallycomprises glucan, xylan, arabinan, mannan, galactan, lignin, ash, andcombinations thereof. Solid phase cellulose fibers are generallyrecovered by solids/liquid separation conducted in accordance withconventional methods known in the art including, for example, utilizinga screen, filter, centrifuge, settler, screw press or belt press. Incertain preferred embodiments, solid phase cellulose fibers arerecovered by filtration of the wet cake.

VIII. Cellulose Hydrolysis and Fermentation

Generally in accordance with the present invention, cellulose issubjected to enzymatic hydrolysis for the primary purpose of hydrolysisof cellulose to produce fermentable sugars. In accordance with theforegoing, cellulose hydrolysis and fermentation feedstock may beprovided by various treatment protocols, and combinations thereof. Forexample, feedstock may comprise biomass that has been subjected topretreatment, conditioning, and xylan hydrolysis in accordance with theforegoing discussion. In various embodiments, the feedstock comprisesbiomass that has been subjected to pretreatment and directly thereaftersubjected to enzymatic hydrolysis of cellulose without intermediateconditioning and/or xylanase treatment. That is, pretreated feedstock 45shown in FIG. 1 is subjected to enzymatic hydrolysis for the primarypurpose of hydrolysis of cellulose to fermentable sugars. Regardless ofthe precise combination of stages prior to enzymatic hydrolysis ofcellulose, the feedstock is generally in the form of a slurry, or cakecomprising a solid fraction or phase comprising cellulose or lignin.

In accordance with the process of FIG. 1, for enzymatic hydrolysis ofcellulose, wet cake 117 is generally contacted with a cellulase enzymeand a liquid medium (e.g., water). Cellulases are a class of enzymesproduced chiefly by fungi, bacteria, and protozoans that catalyze thehydrolysis of cellulose (cellulolysis) into glucose, cellobiose,cellotriose, cellotetrose, and longer chain cellodextrins. Cellulaseincludes both exohydrolysase and endohydrolysases that are capable ofrecognizing cellulose, or cellodextrins, as substrates. Cellulaseenzymes may include endoglucanases, cellobiohydrolysases,beta-glucosidases, alone or in combination.

Conversion of cellulose to fermentable sugars (e.g., six-carbon sugarssuch as glucose) by enzymatic hydrolysis is referred to assaccharification. Sugars produced by saccharification are then fermentedto produce ethanol by contact of the fermentable sugars and yeast orother suitable fermenting organism(s). In accordance with the presentinvention, enzymatic hydrolysis of cellulose may be conducted inaccordance with methods known in the art. For example, the time,temperature, and pH of the saccharification are not narrowly criticaland may generally fall within well-recognized limits. Typically,enzymatic hydrolysis of cellulose is conducted under ambient pressureconditions and at a temperature of from about 20° C. to about 80° C.and, more typically, from about 30° C. to about 60° C.

As shown in FIG. 1, wet cake 117 and enzyme 121 are introduced intocellulose hydrolysis vessel 125 along with water stream 129. Thecellulose content of the cake is typically from about 55 to about 80 wt% (dry weight basis), more typically from about 60 to about 80 wt % and,still more typically, from about 65 to about 75 wt %. The initial solidsloading introduced into the cellulose hydrolysis vessel is generally atleast about 10 wt %, at least about 15 wt %, or at least about 20 wt %.Typically, the initial solids loading introduced into the reactor isfrom about 10 wt % to about 30 wt % and, more typically, from about 15wt % to about 25 wt %.

Generally, the mass ratio of water to wet cake solids introduced intothe hydrolysis vessel and/or a hydrolysis zone therein is at least about1.5:1, at least about 1.8:1, or at least about 2.1:1. Typically, themass ratio of water to wet cake introduced into the hydrolysis vesseland/or a hydrolysis zone is from about 1.5:1 to about 3:1, moretypically from about 1.8:1 to about 2.7:1 and, even more typically, fromabout 2:1 to about 2.5:1.

One measure of the effectiveness of pretreatment is the proportion ofenzyme required to provide suitable fermentable sugar yields. Due to thecost of the enzyme, preferably pretreatment increases thebioavailability of cellulose in a manner that allows for use of arelatively low proportion of enzyme. In accordance with the presentinvention, enzymatic hydrolysis of cellulose may be conducted at enzymeloadings of no more than about 40 FPU per g cellulose, no more thanabout 30 FPU per g cellulose, or no more than about 25 FPU per gcellulose. Typically, the enzyme loading is within the range of fromabout 2 to about 20 FPU per g cellulose, more typically from about 4 toabout 15 FPU per g cellulose and, still more typically, from about 5 toabout 10 FPU per g cellulose. Generally, the mass ratio of enzyme to drycake introduced into the hydrolysis vessel and/or a hydrolysis zone isat least about 0.005, at least about 0.01, or at least about 0.02.Typically, the mass ratio of enzyme to wet cake is from about 0.007 toabout 0.1, more typically from about 0.008 to about 0.08 and, still moretypically, from about 0.01 to about 0.05.

Wet cake (i.e., cellulose), cellulase enzyme, and water are generallycontacted within the hydrolysis vessel and/or a hydrolysis zone at atemperature of from about 25° C. to about 65° C., or from about 35° C.to about 50° C. The duration of contact is typically from about 12 toabout 168 hours, more typically from about 24 to about 120 hours and,still more typically, from about 48 to about 96 hours.

Contacting cellulose, cellulase enzyme, and water yields a cellulosehydrolyzate comprising cellulose-derived sugars (i.e., a C₆hydrolyzate). These include, for example, glucose, dextrose, fructose,and levulose. Generally, the total yield of C₆ sugars in the hydrolyzate(based on the total polysaccharide content of the wet cake introducedinto the hydrolysis vessel) is at least about 50%, at least about 60%,or at least about 70%. Preferably, the total yield of C₆ sugars is atleast about 80% and, more preferably, at least about 90% (e.g., about95%). Typically, the total solids content of the hydrolyzate is fromabout 10 wt % to about 40 wt % and, more typically, from about 20 wt %to about 30 wt %. Typically, the mass ratio of soluble solids toinsoluble solids (e.g., cellulose, glycan, and cellulase enzyme) is fromabout 0.8:1 to about 1.2:1 and, more typically, from about 0.9:1 toabout 1.1:1 (e.g., about 1:1).

Glucose and other fermentable sugars produced by saccharification maythen be fermented to produce ethanol in accordance with methods known inthe art. Again with reference to FIG. 1, cellulose hydrolyzate 133 isremoved from hydrolysis vessel 125 and is introduced into simultaneoussaccharification and fermentation (SSF) vessel 137 for further sugarformation and conversion of sugars to ethanol. SSF is generallyconducted in accordance with conventional methods known in the artincluding, for example, as described in Dowe et al., “SSF ExperimentalProtocols—Lignocellulosic Biomass Hydrolysis And Fermentation”, NationalRenewable Energy Laboratory, 2001, 18 pages, the entire contents ofwhich are incorporated herein by reference for all relevant purposes.The configuration of the SSF reactor is not narrowly critical and may bereadily selected by one skilled in the art. Preferably, the SSF reactorsare suitable for batch or continuous operation (e.g., individual or aseries of continuous stirred-tank reactors).

Further in accordance with the present invention, conversion ofcellulose to fermentable sugars is conducted solely via SSF. Inaccordance with such embodiments, wet cake 117 is introduced into SSFvessel 137 for conversion of cellulose to fermentable sugars. Operationof the process in this manner without a separate cellulose hydrolysisstep provides a process of reduced cost. However, while utilizing SSFalone for generation of fermentable sugars may provide improvements inprocess economies, it is to be understood that the process depicted inFIG. 1 can be practiced in an economical manner.

Along with cellulose (i.e., C₆) hydrolyzate 133, yeast inoculum 141 isintroduced into SSF vessel 137. Suitable yeast include those noted aboveand, in various preferred embodiments, the yeast is Sacchromycescerevisiae. Yeast inoculum 141 introduced into the SSF vessel comprisesthe yeast dispersed throughout an aqueous medium. Typically, the yeastcontent of the yeast inoculum is from about 0.1 to about 5 wt % and,more typically, from about 1 to about 2.5 wt %. The relative proportionsof yeast inoculum and cellulose hydrolyzate introduced into the SSFvessel depend on a variety of factors including, for example, thecomposition of each stream. Generally, however, the mass ratio of yeastinoculum to hydrolyzate introduced into the SSF vessel is from about0.01:1 to about 0.25:1, or from about 0.02:1 to about 0.1:1. Althoughnot narrowly critical, preferably saccharification and fermentation arecomplete after a period of operation of no more than about 168 hours, nomore than about 144 hours, or no more than about 96 hours.

Contacting cellulose hydrolyzate 133 and yeast inoculum 141 within SSFvessel 137 yields a C₆ fermentate 145. Generally, the C₆ fermentate is amixture of water, ethanol, and unconverted sugars and fibers (e.g.,carbohydrate, lignin, and ash) of the enzymatic hydrolyzate. The overallcomposition of the C₆ fermentate generally varies depending on, forexample, the composition of the enzymatic hydrolyzate, yeast inoculum,and the relative proportions introduced into the SSF vessel. Preferably,the composition of the C₆ fermentate represents suitable yields ofethanol based on the fermentable sugar content of the enzymatichydrolyzate. For example, generally the ethanol yield of the C₆fermentate is at least about 20%, at least about 30%, or at least about40%. It is currently believed that ethanol yields satisfying theselimits, and higher, are achieved in accordance with the process depictedin FIG. 1. Typically, the concentration of ethanol in the C₆ fermentateis at least about 2 wt %, more typically at least about 4 wt % and,still more typically, at least about 5 wt %. The residual fermentablesugar content of the C₆ fermentate also depends on a variety of factorsincluding, for example, the composition of the enzymatic hydrolyzate andethanol yield achieved. Typically, however, the residual fermentablesugar content of the C₆ fermentate is less than about 5 g/L, moretypically less than about 3 g/L and, still more typically less thanabout 2 g/L. Unconverted C₆ sugars may be converted to biogas during theanaerobic digestion step of wastewater treatment and/or concentrated andcombusted in a biomass boiler.

IX. Ethanol Recovery

Again with reference to FIG. 1, C₆ fermentate 145 is introduced into astill 149 into which steam 155 is introduced wherein the fermentate isdistilled to produce a high wines fraction 153 derived from C6 sugars(or C6 and C5 sugars) and a bottoms product 157. Distillation generallyproceeds in accordance with conventional methods known in the art usingconventional apparatus as described, for example, in DistillationTechnology, GEA Wiegand, 16 pages and Bioethanol Technology, GEAWiegand, 16 pages, the entire contents of which are incorporated hereinby reference for all relevant purposes. The high wines fraction may thenbe dehydrated to produce ethanol product. Generally, conventionaldistillation apparatus known in the art are suitable for use inaccordance with the present invention. These include, for example,distillation columns including dual flow and cross flow trays. However,because of the high suspended solids content of the fermentate, or beerstream, generally dual flow sieve trays or cross-flow valve trays arepreferred. In various preferred embodiments, columns including crossflow valve trays are preferred because of the higher turn down ratio andhigher efficiency often provided by cross flow valve trays. Suitablevalve trays include, for example, NORPRO PROVALVE trays.

As detailed herein, various strategies of the present inventionpreferably maximize ethanol yields. For example, in various preferredembodiments of the present invention ethanol yields of at least about70%, at least about 75%, or at least about 80% (basis total celluloseand hemicellulose content of feedstock) may be achieved.

X. Ethanol Co-Products

Ethanol distillation bottoms product 157 is generally in the form of aslurry, or cake comprising solid remnants of the feedstock. The bottomsproduct may be separated (e.g., by centrifugation) to produce highsolids distiller's grains 161 and thin stillage 165.

The distiller's grains 161 may be dried to produce a solid proteinproduct. For example, dried distiller's grains having a protein contentof at least about 10 wt %, at least about 15 wt %, or at least about 20wt % may be prepared upon drying of the distiller's grains.

Thin stillage 165 is generally in the form of an aqueous waste streamhaving total solids content of no more than about 2 wt %, and preferablyno more than about 1 wt %. Accordingly, thin stillage may be subjectedto treatment prior to disposal (not shown in FIG. 1) and/or may beutilized as process water (also not shown in FIG. 1). For example, asnoted, thin stillage may provide at least a portion of the process waterutilized during conditioning and/or sugar extraction as detailedelsewhere herein.

XI. Integrated Cellulase Generation

Further in accordance with the present invention, enzyme for use inenzymatic hydrolysis may be prepared utilizing a portion of feedstock.As shown in FIG. 1, a portion of the wet cake 117 is introduced intovessel 118 along with supplement stream 119 for production of enzyme.

Supplement 119 generally comprises an aqueous biosynthesis medium,nitrogen and/or nutrient source, and a microbe that is effective toexpress a cellulase enzyme.

Suitable biosynthesis media include water, sugars, nutrients, andcombinations thereof. In various preferred embodiments, the biosynthesismedium is water. Suitable sugar, nitrogen and/or nutrient sourcesinclude corn syrup, molasses, cereal mash and distiller's dried grainsremaining after recovery of ethanol. Nutrients present in the supplementinclude, for example, calcium, phosphorus, potassium, magnesium, iron,manganese, zinc, and combinations thereof. Suitable microbes includeTrichoderma reseei, Aspergilus, and combinations thereof. Supplement 119also typically comprises glucose that can be utilized by the microbe forformation thereof.

Typically, the glucose content of supplement 119 is from about 10 wt %to about 50 wt % and, more typically, from about 20 wt % to about 40 wt%. The nitrogen and/or nutrient source typically constitutes from about1 wt % to about 20 wt % and, more typically, from about 5 wt % to about15 wt % of the supplement. However, the precise composition of thesupplement is not narrowly critical. Substrates suitable for enzymeproduction are detailed in Example 4. Generally, the substrates providea carbon source, nitrogen source, and nutrients for growing theenzyme-producing microorganisms. For example, suitable carbon sourcesinclude glucose syrups (e.g., having a glucose content of 75% orgreater), pretreated (and preferably washed) biomass, cereal mash, anddistiller's dry grains with solubles (recovered as detailed elsewhereherein). In various preferred embodiments the substrates comprise fromabout 45 to about 65 wt % (preferably about 55 wt %) corn syrup, fromabout 10 to about 20 wt % (preferably 15 wt %) washed and pretreatedbiomass, from about 10 to about 20 wt % (preferably about 15 wt %)cereal mash, and from about 10 to about 20 wt % (preferably about 15 wt%) distiller's dry grains with solubles. Suitable nitrogen sourcesinclude urea, ammonium hydroxide, ammonium sulfate, and combinationsthereof Suitable nutrients include corn syrup liquor, inorganic salts(e.g., containing magnesium, potassium, calcium, phosphate, iron, andmanganese) and combinations thereof.

Vessel 118 generally comprises a microbe proliferation zone in whichglucose, cellulose, the nitrogen and/or nutrient source, and the microbeare contacted. The mass ratio of supplement to the portion of the wetcake introduced into the vessel and/or contacted within the microbeproliferation zone is typically from about 1:1 to about 10:1 and, moretypically, from about 2:1 to about 8:1. Generally, from about 0.1 toabout 5 wt %, or from about 0.5 to about 2.5 wt % of the wet cake isintroduced into the vessel and/or contacted with the supplement withinmicrobe proliferation zone. Generally, the portion of the aqueous wetcake and the supplement are contacted at a temperature of from about 20°C. to about 60° C. and, more typically, at a temperature of from about30° C. to about 50° C.

Contacting glucose, cellulose, the nitrogen/nutrient source, and themicrobe within the proliferation zone of vessel 118 yields an enzymeslurry comprising a solid enzyme fraction and a liquid fraction. Thesolid enzyme fraction typically constitutes from about 1 to about 15 wt% and, more typically, from about 5 to about 10 wt % of the enzymeslurry. The remainder of the enzyme slurry generally comprises water. Asshown in FIG. 1, enzyme 121 is introduced into cellulose hydrolysisvessel 125 along with wet cake 117.

XII. Yeast Preparation

Again with reference to FIG. 1, yeast inoculum 141 is prepared in yeastpropagation vessel 138 by combining yeast supplement 139 and yeast 140.As noted, suitable yeast for use in the simultaneous saccharificationand fermentation include Sacchromyces cerivisiae. Yeast supplement 139generally comprises Sacchromyces cerivisiae, and in accordance with theembodiment depicted in FIG. 1, is a glucose syrup, or slurry, comprisingglucose dispersed throughout an aqueous medium (e.g., water). Moreparticularly, and in accordance with various preferred embodiments,yeast supplement 139 is typically a glucose syrup containing glucose ina proportion of at least about 5 wt % glucose, more typically at leastabout 10 wt % glucose. The conditions of yeast propagation are notnarrowly critical and propagation is generally conducted in accordancewith conventional methods known in the art including, for example, ScottLaboratories, Yeast Rehydration Protocol, 1 page and Propax YeastPropagation Technology, Meura, 2 pages, the entire contents of which areincorporated herein by reference for all relevant purposes.

XIII. FIG. 3

FIG. 3 depicts another embodiment of a process of the present invention.Generally, preparation of pretreated feedstock, and preparation andrecovery of a C₅ hemicellulose-derived sugar fraction proceeds inaccordance with the discussion above regarding FIG. 1. Accordingly, thedescription of these steps of the process depicted in FIG. 3 will not berepeated. With reference to FIG. 3, recovery of a C₅ sugar fractionyields a residual thickened fraction 85A in the form of a cake orconcentrated slurry comprising solid phase cellulose and lignin (i.e.,cellulose/lignin residual fraction). Contrary to the process depicted inFIG. 1, residual thickened fraction 85A is not introduced into a vesselfor extraction of lignin but, rather, a portion of the residualthickened fraction 85A is introduced into enzyme production vessel 118Awhile another portion is introduced into cellulose hydrolysis vessel125A. Typically, a minor portion (e.g., less than about 5 wt %, lessthan about 2 wt %, or less than about 1 wt %) of the residual thickenedfraction 85A is introduced into enzyme production vessel 118A. Thecomposition of residual thickened fraction 85A generally corresponds tothe composition of residual thickened fraction 85 discussed above inconnection with FIG. 1.

Again with reference to FIG. 3, cellulose hydrolysis within cellulosehydrolysis vessel 125A, SSF within vessel 137A, and distillation withinstill 149A generally proceed in accordance with the discussion set forthabove regarding the process of FIG. 1. However, certain adjustments maybe made based on the varied composition of the cellulose/lignin residualfraction introduced into cellulose hydrolysis vessel 125A. For example,generally in accordance with the process of FIG. 3 an increasedproportion of residual fraction is introduced into the cellulosehydrolysis vessel as compared to the process of FIG. 1. Thus, anincreased proportion of water is typically introduced into the cellulosehydrolysis vessel.

Further in accordance with the process depicted in FIG. 3, distillationyields a high wines fraction 153A derived from C6 sugars (or C6 and C5sugars) and a bottoms product 157A. Bottoms product 157A is separated(e.g., by centrifugation) to produce high solids distiller's grains 161Aand thin stillage 165A.

As noted above in connection with the process depicted in FIG. 1, thedistiller's grains are generally rich in protein derived from theinitial protein content of the biomass and based on protein generatedduring the process (e.g., during integrated enzyme generation). Inaddition, in accordance with the process depicted in FIG. 3, asignificant fraction of lignin remains in the distillation bottomsproduct since lignin fractionation does not occur prior to derivation offermentable sugars by enzymatic hydrolysis of hemicellulose andcellulose and fermentation of the sugars. The lignin content of thedistiller's grains 161A is generally from about 30 to about 60 wt %, andtypically from about 40 to about 60 wt %.

Contrary to the process depicted in FIG. 1, in accordance with theprocess depicted in FIG. 3, a lignin-rich product is not recovered priorto enzymatic hydrolysis of cellulose to fermentable sugars or productionof ethanol therefrom. Instead, insoluble lignin and lignin productsremain in the distiller's solid residue or cake (i.e., dry grains) 161A.As shown in FIG. 3, distiller's solid residue 161A is introduced intolignin extraction vessel 170A along with an extraction solvent 175A. Thecomposition of the extraction solvent is not narrowly critical, butgenerally is in the form of the extraction solvents described above. Invarious preferred embodiments, the extraction solvent is in the form ofan aqueous solution of sodium hydroxide. Typically, the distiller'ssolid residue and extraction solvent are contacted at a temperature offrom about 30° C. to about 60° C. and, more typically, from about 40° C.to about 50° C. (e.g., about 45° C.).

Mixing the distiller's solid residue and extraction solvent within anextraction zone of the extraction vessel forms an extraction mixturecomprising an extract comprising lignin and a wet cake. Again withreference to FIG. 3, lignin extract 180A and lignin wet cake 185A areremoved from the lignin extraction vessel.

The lignin extract comprises a solids fraction and a liquid fraction andtypically has a total solids content of from about 1 to about 15 wt %,and more typically from about 2.5 to about 10 wt %. Lignin typicallyconstitutes at least about 1 wt %, more typically at least about 2 wt %and, still more typically, at least about 3 wt % of the lignin extract.For example, lignin generally constitutes from about 1 to about 10 wt %,or from about 2 to about 6 wt % of the lignin extract. Lignin wet cakeis generally in the form of a slurry containing up to 25 wt % or up to30 wt % solids content and various impurities. Preferably, and inaccordance with the embodiment depicted in FIG. 3, the wet cake does notcontain lignin for recovery and, accordingly, is generally removed fromthe process as a waste stream.

A lignin-rich product may be recovered from lignin extract 180Agenerally as described above in connection with lignin extraction vessel113A utilized in the process of FIG. 1. The lignin-rich product(typically in the form of a powder) generally exhibits any or all of theproperties noted above in connection with the lignin-rich productprovided by the process of FIG. 1.

Optionally (as indicated by the dashed line in FIG. 3), a lignin extractportion 190A may be recovered from lignin extraction vessel 170A andintroduced into evaporator 195A for removal of moisture to form alignin-rich product 200A. Lignin-rich product prepared by evaporation isgenerally in the form of a slurry of lignin-rich solids having a totalsolids content of from about 20 wt % to about 50 wt %, or from about 20wt % to about 40 wt %. The lignin-rich slurry product may be utilizedas-is in a variety of applications (e.g., wood composite adhesive) ormay be further processed to provide a lignin-rich product of greaterpurity, or a dry powder comprising lignin monomers or other degradationproducts (not shown).

As further shown in FIG. 3, lignin extract 180A may be introduced into aprecipitation vessel 181A where it is contacted with an acid 182Asuitable for forming lignin precipitate 183A. Lignin precipitate 183A isfiltered and dried in a suitable vessel 184A to form a lignin powderproduct 186A and lignin waste stream 187A. Lignin waste stream 187A maybe removed as a waste water stream and sent for wastewater treatment.

XIV. Recovery of Heat Values

Further in accordance with the present invention, one or more processstreams or residues may be introduced into a biomass boiler for recoveryof heat values from organic components of the stream, or residue. Theheat values thus recovered may be utilized for steam generation. Inparticular, heat values may be recovered from carbohydrates (e.g.,unconverted C₅ and C₆ sugars) by combustion in a biomass boiler. Forexample, and again with reference to FIG. 1, distillation bottomsproduct 157 may be sent to a biomass boiler for combustion and recoveryof heat values therefrom. Again with reference to FIG. 1, thedistiller's solid residue 161 typically has a solids content of fromabout 30 to about 40 wt % (e.g., from about 32 wt % to about 38 wt % orfrom about 34 wt % to about 36 wt %). Thus, various alternativeembodiments include sending the distiller's solid residue to a biomassboiler for recovery of heat values. The heat value (energy content) ofthe solid residue is typically from about 7,000 to about 8,500 BritishThermal Units (BTU) per pound (lb) (dry weight basis) (BTU/lb).Additionally or alternatively, thin stillage 165 may be utilized forrecovery of heat values from its organic components. As noted, typicallythin stillage 165 has a total solids content of no more than about 5 wt% (e.g., no more than about 2 wt %). Accordingly, prior to introductioninto the biomass boiler, the thin stillage may be subjected toevaporation to provide a feed for the biomass boiler having a suitablesolids content (e.g., from about 50 to about 70 wt %). The heat value(energy content) of the concentrated thin stillage is typically fromabout 5,000 to about 6,500 BTU/lb.

Further in accordance with the present invention and with reference toFIG. 3, thin stillage 165A and/or lignin waste stream 187A may subjectedto evaporation to provide a feed for the biomass boiler having asuitable solids content (e.g., from about 50 to about 70 wt %). Thetypical heat content of such a concentrated waste stream is typicallyfrom about 5,000 to about 6,500 BTU/lb.

The present invention is illustrated by the following examples which aremerely for the purpose of illustration and not to be regarded aslimiting the scope of the invention or the manner in which it may bepracticed.

EXAMPLE 1

This example details acid impregnation of corn stover (CS) harvestednear Hugoton, Kans. A bale of corn stover weighing approximately 700pounds was manually de-stringed and introduced into a tub grinder(Vermeer Corporation, Pella, Iowa, U.S.A., Model TG 7000) including ascreen having openings of approximately 3 inches to provide coarselymilled feedstock. The coarsely milled corn stover was then milledthrough a 0.5-inch screen using a hammer mill (Bliss Industrial Inc.,Ponca City, Okla. U.S.A., Model ER-2215-TF. The milled corn stover had amoisture content of approximately 11 wt %.

Following milling, 20 lb batches of the 0.5 inch milled corn stover wereimpregnated with sulfuric acid. For acid impregnation, the batches ofcorn stover were tumbled in a 70° C. jacketed double-shaft mixer whileapproximately 20 lb of a 3% (w/w) solution of sulfuric acid at atemperature of 70° C. was sprayed onto the milled feedstock for a periodof 2 minutes. After spraying was complete, the acid-feedstock mixturewas mixed for an additional 6 minutes. The resulting acid-impregnatedcorn stover was then held in a 70° C. jacketed surge bin beforepretreatment for a hold time of from approximately 20 minutes.

Acid-impregnated corn stover was then introduced into a pre-heated batchdigester having a total volume of approximately 100 liters (l). Steamunder a pressure of 200 psig was introduced into the digester forheating of the corn stover for approximately 10 to 30 seconds. Duringthe first 5 to 10 seconds of the steam injection period, a 0.5 inch ventvalve on top of the digester was opened to purge air from the digester.After steam injection was completed, the acid-impregnated feedstock washeld in the digester under a steam pressure of approximately 200 psigfor approximately 130 seconds after which time the feedstock wasdischarged from the digester under a pressure of 200 psig. Discharge ofthe feedstock occurred over a vent time of approximately 20 seconds.

For comparison purposes, pretreatment was tested in which the feedstockwas discharged under pressures below 200 psig in which the pressure wasreduced to pressures of approximately 150 psig, 120 psig, and 100 psigby venting of the digester for approximately 20 seconds. For comparisonpurposes, pretreatment was tested in which the feedstock was subjectedto first and second stages of different pressure conditions. The firststage occurred over a period of approximately 1 to 5 minutes duringwhich time the feedstock was subjected to pressures of fromapproximately 150 to approximately 230 psig. After the first stage wascompleted, the digester was vented for approximately 10 to 30 seconds toreduce the pressure in the digester by approximately 50 to 150 psig orapproximately 75 to 120 psig. The duration of the second stagepretreatment was approximately 0.2 to 5 minutes or approximately 0.5 to3 minutes.

Samples from each batch of pretreated corn stover (PCS) were analyzedfor chemical composition.

Table 1 provides the composition of the liquor in samples of corn stoverpretreated at the various pressures. The moisture content of the PCSsamples are not the same, with variations up to 10%. The sugarconcentrations in the liquors are normalized to the moisture content asthe PCS sample of the full pressure pretreatment (i.e., single-step, 200psig pretreatment) for direct comparison.

TABLE 1 Discharge Pressure 200 psi 150 psi 120 psi 100 psi Concentrationin Before acid After 4% acid Before acid After 4% acid Before acid After4% acid Before acid After 4% acid liquor, g/L hydrolysis hydrolysishydrolysis hydrolysis hydrolysis hydrolysis hydrolysis hydrolysisGlucose 20.2 24.16 19.94 23.23 20.18 23.67 23.96 27.85 Xylose 79.9988.79 85.10 91.66 89.44 97.63 92.63 101.60 Galactose 6.6 8.06 8.46 9.526.91 8.30 5.51 7.31 Arabinose 10.69 12.68 11.37 13.57 10.75 13.20 9.3112.05 Mannose 2.23 2.84 2.01 2.71 1.81 2.57 1.54 2.51 Cellobiose 1.79 —2.68 — 1.92 — 2.07 — Acetic Acid 4.77 — 4.73 — 5.78 — 5.23 — Furfural1.54 — 1.82 — 2.03 — 2.47 — HMF 0.87 — 0.90 — 0.71 — 1.05 — Totalsoluble sugars 121.5 136.53 129.55 140.67 131.00 145.38 135.03 151.32

Table 2 shows the composition of washed corn stover pretreated at thevarious pressures.

Table 3 shows the results of enzymatic hydrolysis of washed andpretreated samples.

Table 4 shows the particle size analysis of washed PCS.

TABLE 2 Discharge pressure Component, wt % 200 psi 150 psi 120 psi 100psi Glucan 55.96 56.32 56.47 55.47 Xylan 4.03 3.64 2.94 2.8 Galactan 0.30.3 0 0 Arabinan 0.86 0.88 0.77 0.67 Mannan 0.35 0.33 0 0 Klason Lignin26.82 27.7 28.94 29.82 Acid soluble lignin 1.13 1.08 1.06 0.99 Ash 9.2810.26 10.18 11.03 Acetic acid 0.77 0 0 0 Mass balance 99.50 100.52100.35 100.78

TABLE 3 Enzyme hydrolysis Discharge pressure time, hr 200 psi 150 psi120 psi 100 psi 0 0.0 0.0 0.0 0.0 12 45.2 42.8 43.0 44.7 24 64.9 61.862.9 62.9 48 85.1 81.9 84.7 83.8 72 90.5 90.2 93.5 88.8 96 92.9 92.995.8 91.2

TABLE 4 Arithmetic Weighted Fine elements Fine elements CoarsenessDischarge Pressure, psi length (μm) length (μm) Width (μm) (% in length)(% in area) (mg/m) 200 181 303 23.3 41.3 2.87 0.2283 150 179 291 23.441.4 2.73 0.2476 150 (unwashed PCS) 177 291 23.3 41.3 2.71 0.2421 100159 248 23.8 50.3 3.22 0.2455

These results indicate that two-step pretreatment provided an increaseof up to approximately 11% in soluble sugar yield in comparison with thesingle-step pretreatment. It is believed that a significant portion, ifnot most of the increase can be attributable to additional xylanconversion as evidenced by the increase in xylose concentration in theliquor and reduction in xylan content of washed pretreated corn stover.It is currently believed that venting furfural during pretreatmentprevents furfural from condensing on xylose, thereby leading to highersolubilized xylose yield. It is further currently believed thatadvantageous ethanol yield is provided by virtue of recovery andfermentation of C₅ sugars. Particle size and cellulose digestibilityvaried little between corn stover pretreated by one and two stepmethods.

EXAMPLE 2

This example details power input required during acid impregnationcarried out as described in Example 1.

Power input ranged from 1.7 to 8.5 kWh/ton of corn stover for 2 to 10minutes mixing time. (Mixing power input =mixing power with straw andacid minus mixing power without corn stover). Acid impregnation mixingtime ranged from 4 to 6 minutes, which corresponds to a power input offrom 3.4 to 5.1 kWh/ton corn stover (dry weight basis). The moisturecontent of the milled corn stover was 12 wt % and the moisture contentof the acid-impregnated corn stover was approximately 55 wt %. Tables 5and 6 provide the results for the power input testing wherein, in Table5, the voltage for each Empty, CS only and CS plus acid solutionoperation was 460 V, the run time for each Empty, CS only and CS plusacid solution operation was 6 minutes, the corn stover (CS) input as isfor each Empty, CS only and CS plus acid solution operation was 20 lb,the dry weight of CS for each Empty, CS only and CS plus acid solutionoperation was 17.6 lb, and the total solids (TS) of CS was 0.88 for eachEmpty, CS only and CS plus acid solution operation.

TABLE 5 Estimation of acid impregnation power consumption Power inputPower input Current, Power consumed per batch of per ton CS, OperationAmp kVA kW per batch, kWh CS, kWh kWh Empty 1.49 1.1871 0.9497 0.09500.0000 N/A CS only 1.85 1.4739 1.1791 0.1179 0.0229 2.607 CS plus acid2.19 1.7448 1.3959 0.1396 0.0446 5.070 solution Impregnator run time,min kWh for wet CS 2 1.69 Normal run = 6 min 3 2.53 4 3.38 5 4.225 65.07 7 5.915 8 6.76 9 7.605 10 8.45 kVA = V * A * 1.732/1000 (Note:1.732 is square root of 3) kW = kVA * Power Factor (Note: Power factor(PF) for motors 1-5 HP PF = 0.75, 5-50 HP motors PF = 0.8, 50-100 HP, PF= 0.85, >100 HP PF = 0.9) Weight of CS per batch (20 lb)

EXAMPLE 3

This example provides results of particle size analysis of corn stoverpretreated by a variety of combinations of conditions.

Table 6 provides the results of particle size analysis of milled cornstover using the specified U.S. Standard Sieves. Generally, the particlesize distribution of pretreated corn stover is narrower than that ofmilled corn stover.

TABLE 6 Average Standard Sieve Opening, mm/μm retained % Cumulative, %Deviation Tray 0.265″ 6.73 mm 0.0 na 0.0 Tray #5 4 mm 10.8 100 0.7 Tray#10 1.68 mm 29.2 89.2 1.5 Tray #20 841 μm 27.7 60 1.4 Tray #40 420 μm14.6 32.3 0.6 Tray #60 250 μm 4.9 17.7 0.8 Bottom <250 μm 12.8 12.8 1.1

Particle size analysis was conducted for (a) corn stover pretreated atvarious pressures; (b) water washed corn stover, and (c) fiber recoveredfrom pretreated corn stover stillage. Generally, as described below, atreduced-pressure blow (100 psig vs full-pressure blow at 200 psig), thecoarseness increases slightly. Accurately measuring the fiber length forsmall particles using the Fiber Quality Analyzer proved to be difficult.

Particle size data were collected using a Fiber Quality Analyzer(FQA)—MorFi OL-01 commercially available from Techpap (France). FiberLength and Fiber Width measurements were taken. For comparison purposes,particle size analysis of wheat straw was also conducted. Results areprovided in Table 6.

Fiber Length: Arithmetic average fiber length and length weightedaverage fiber length. Arithmetic average fiber length is the sum of allthe individual fiber lengths divided by the total number of fibersmeasured; Length weighted average fiber length is calculated as the sumof individual fiber lengths squared divided by the sum of the individualfiber length. The data in Table 6 indicate (a) as pretreatment pressureincreases fiber length increases; (b) water washing does not affect thefiber length; (c) fiber length of pretreated corn stover stillage andpretreated wheat straw are similar.

Fiber Width: measurement of length across the fiber. The results listedin Table 6 indicate (a) higher pretreatment pressure generally providesshorter fiber width up to 150 psig, after which no effect on fiber widthwas observed; (b) water washing does not affect fiber width; (c) thefiber width from pretreated corn stover stillage is larger than one frompretreated wheat straw stillage.

Coarseness: milligrams of fiber per meter of fiber length. The resultsin Table 6 indicate (a) a maximum value for coarseness (0.2476) atpretreatment pressure of 150 psig; (b) water washing process increasesthe fiber coarseness; (c) the coarseness from pretreated corn stoverstillage is much bigger than the one from pretreated wheat strawstillage.

Fines: particles below 7 microns (above 7 microns a “fiber”). Thepercentage of fines on an arithmetic basis is the number of finesdivided by the total number of fibers (fines included) multiplied by100%; the percentage of fines on a length weighted basis is the sum offines length divided by the total length of fibers and fines in thesample.

The results in Table 7 indicate (a) higher pretreatment pressuregenerally results in less fines up to 150 psig pressure, but above 150psig pressure does not affect the proportion of fines on a lengthweighted basis; based on the number of fines (2.73) there is a minimumat a pressure of 150 psig; (b) water washing does not affect finecontent; (c) pretreated corn stover stillage provides a higherproportion of fines than pretreated wheat straw stillage; (d) stillage(particularly from pretreated corn stover) includes a relatively highproportion of fines (e.g., over 95%), which raises issues duringfiltering during water washing.

TABLE 7 Tabel 1. Fiber properties from seven (7) samples by FQAArithmetic Weighted Width Fine Elements Percentage of Coarseness Length(μm) Length (μm) (μm) (% in length) fine elts (% in area) (mg/m) Sample1 2 Ave. 1 2 Ave. 1 2 Ave. 1 2 Ave. 1 2 Ave. 1 2 Ave. WW-PCS- 180 182181 300 305 302.5 23.2 23.4 23.3 41.3 40.5 41.3 2.94 2.79 2.865 0.23360.223 0.2283 200 Pisg WW-PCS- 178 179 178.5 290 291 290.5 23.5 23.3 23.441.4 40.4 41.4 2.79 2.67 2.73 0.236 0.2591 0.2476 150 Pisg WW-PCS- 160158 159 246 249 247.5 23.9 23.7 23.8 50.3 50.1 50.3 3.17 3.27 3.220.2385 0.2524 0.2455 100 Pisg WW-PCS- 178 179 178.5 290 291 290.5 23.523.3 23.4 41.4 40.4 41.4 2.79 2.67 2.73 0.236 0.2591 0.2476 150 PisgPCS- 175 179 177 289 292 290.5 23.4 23.2 23.3 41.3 40.5 41.3 2.73 2.682.705 0.2423 0.2418 0.2421 150 Pisg PCS- 110 111 110.5 130 132 131 2323.4 23.2 95.8 95.6 95.8 11.21 11.1 11.15 0.9574 0.9656 0.9615 StillagePCS- 114 114 114 134 132 133 24.3 24.4 24.35 93.8 93.8 93.8 9.66 9.659.655 1.3279 1.3262 1.3271 SSF residue PCS- 110 111 110.5 130 132 131 2323.4 23.2 95.8 95.6 95.8 11.21 11.1 11.15 0.9574 0.9656 0.9615 StillagePWS- 111 112 111.5 128 131 129.5 21.3 21.5 21.4 87.2 86.8 87.2 7.35 7.127.235 0.4955 0.4874 0.4915 Stillage

EXAMPLE 4

This example details a suitable method for preparation of a cellulaseenzyme. Produce enzymes for the saccharification of pretreated biomassby a genetically modified microorganism expressing high levels of themain enzymatic activities required for cellulose hydrolysis. Grow themicroorganism from laboratory cultures through bioreactors of increasingvolume (pre-seed and seed propagation fermentors) in order to prepare aninoculum of approximately 10% of the volume of the production fermentorin the scheduled time of approximately 144 hours.

Enzyme production generally includes (1) media preparation and (2)fermentation. Media preparation includes substrate preparation andnutrient preparation steps.

Utilize 75% glucose syrup, washed pretreated biomass, cereal mash anddistiller's dry grain with solubles as substrates. These substratesgenerally provide a carbon source for growing the enzyme-producingmicroorganisms. The glucose syrup provides a high concentration ofglucose that can be readily utilized by the microorganism withouthindering mass transfer in the fermentation process. The washedpretreated biomass is a source of cellulose that enhances the celluloseactivity of the enzymes produced towards the substrate to be hydrolyzedin the saccharification step. Hydrolyzed cereal mash and distiller's drygrain with solubles (DGS) are low-cost substrates, which are readilyavailable in facilities including biomass ethanol production and cerealbiomass ethanol production. The cereal mash and DGS also providesupplemental nitrogen source and other nutrients (e.g., Ca, P, K, Mg,Fe, Mn, and Zn). Enzyme protein yields of approximately 0.33 g proteinper g of glucan and glucose available in the combined substrates may beachieved. Table 8 provides the composition of a suitable substrate.

TABLE 8 % dry weight of total combined Substrate substrates (dry weightbasis) Corn syrup  55 ± 10 Hydrolyzed cereal mash 15 ± 5 DGS 15 ± 5Washed pretreated biomass 15 ± 5

Blend the substrates together or slurry with the nutrient solutionsbefore adding to the seed and production fermentors. The initialconcentration (dry weight basis) of the combined substrates in thefermentors will typically be about 7% to achieve a C:N ratio of about4:1. Through fed-batch fermentation (e.g., including stepwiseintroduction of substrates to the vessel), the effective substrateloading can be as high as 30% (total initial insoluble and solublesolids).

In addition to substrates described above, the microorganism requiresnitrogen and nutrients for growth and enzyme protein production. Themajor nutrient requirements are nitrogen sources (organic andinorganic). Organic nitrogen is provided from protein contents of mash,DGS, supplemental protein sources such as soybean meal, soy proteinconcentrate, and Pharmamedia (a finely ground yellow flour prepared fromthe embryo of cottonseed; the principle component is nonhydrolyzedglobular protein). The initial crude protein concentration from organicsource in the fermentation step may be about 15 g/L. Inorganic nitrogenmay be supplied at an initial concentration of about 12 g/L via additionof ammonium sulfate ((NH4)₂SO₄). Suitable additional nutrient sources,and their concentration in the fermentors, are listed in Table 9.

TABLE 9 Component¹ Initial concentration in fermentors Lactose² 10-20KH₂PO₄ 3 MgSO₄•7H₂O 0.3 CaCl₂•2H₂O 0.4 FeSO₄•7H₂O 0.005 MnSO₄•H₂O 0.002ZnSO₄•H₂O 0.0014 Yeast extract 0.16 ¹Note: elements may be partially orfully fulfilled by addition of the substrates (such as mash and DGS)²Lactose is added primarily as an enzyme inducer

Combine all nutrients in sterilized make-up process water and store foruse.

Conduct fermentation in successive batch fermentors. Grow enzymecultures of the microorganism in lab fermentors or flasks and transferaseptically into pre-seed fermentors. The pre-seed and seed fermentorsprovide a 10% inoculum for the production fermentors. Each cycle oftransfer and fermentation time may be about 144 hours. Carry outfermentation in fed-batch mode to achieve high enzyme proteinconcentration in the final broth under conditions of: 32±2° C., pH 4.5,air sparging rate of 0.5 vvm (volume of air per volume of broth perminute), over the course of 144 hours (including feeding and removal offermentation broth). Once fermentation is complete, transfer the culturebroth containing the active enzymatic mixture and store in enzymestorage tank(s) which are cooled (at less than approximately 25° C.)utilizing a chilled water jacket.

EXAMPLE 5

This example provides a mass balance (Table 10) for an ethanolproduction process of the present invention prepared using corn stoverand generally corresponding to the process depicted in FIG. 1.

TABLE 10 Milled & Pre- PCS feed cleaned Dilute Acidified treatmentPretreatment Conditioned to sugar Wash CS Acid CS steam flash steam PCSReactants PCS slurry Enzyme extraction water 1 2 3 4 5 6 7 8 9 10 11Component & Units Total Flow kg/hr 46.30 48.38 94.68 27.46 13.61 106.0616.17 122.23 0.95 123.18 80.83 Dry total solids, kg/h 41.67 40.42 1.6242.03 0.07 42.11 Total Solids, wt fraction 0.90 0.38 0.10 0.34 0.08 0.34Moisture, wt fraction 0.10 0.62 0.90 0.66 0.92 0.66 Insoluble, wtfraction 1.00 0.65 0.36 0.64 0.22 Soluble solids, wt 0.00 0.35 0.54 0.360.12 fraction Insoluble, kg/hr 26.27 0.58 26.85 0.07 26.93 Solublesolids, kg/hr 14.15 1.03 15.18 15.18 Temperature ° C. 20.0 60.0 60.0192.0 100.0 50.0 60.0 20.0 60.0 70.0 pH 1.10 1.80 4.50 4.50 4.50 4.506.80 Pressure, psig 14.7 175.0 Steam, kg/hr 27.46 13.61 Water kg/hr 4.6347.44 52.07 65.64 14.55 80.19 0.88 81.07 80.83 Ethanol kg/hr Glucose +oligomers 1.78 (SS) kg/hr Xylose + oligomers 9.22 (SS) kg/hr Arabinose +oligomers 1.30 (SS) kg/hr Non-glucose C6 Sugar + 1.10 oligomers (SS)kg/hr Lignin (SS) kg/hr 0.48 Inorganic Salts (SS) 0.38 kg/hr Volatileorganics (acetic 1.02 acid + furfural + HMF) kg/hr Lactic Acid kg/hrUronic Acid kg/hr 0.64 Ammonia (NH3) kg/hr NaOH kg/h Sulfuric acid kg/hr0.94 0.94 0.94 Carbon Dioxide kg/hr Oxygen kg/hr Nitrogen kg/hr Starch(IS) kg/hr Glucan (IS) kg/hr 16.08 16.08 14.47 Xylan (IS) kg/hr 9.029.02 0.90 Arabinan (IS) kg/hr 1.45 1.45 0.30 Non-glucose C6 Solid 1.221.22 0.23 (IS) kg/hr Lignin (IS) kg/hr 8.00 8.00 7.52 Acetate (IS) kg/hr0.83 0.83 0.04 Uronic Acid (IS) kg/hr 1.29 1.29 0.64 Ash (IS) kg/hr 1.881.88 1.50 Protein kg/hr 1.50 1.50 0.07 Yeast (IS) kg/h Enzyme (IS) kg/hrUnknown (IS) kg/hr 0.40 0.40 0.40 Unknown (SS) kg/hr Corn mash (35% TS),kg/hr 75% glucose syrup Nutrients, kg/hr Sugar extract Suplement XyloseXylose Wet cake Caustic to to xylose 1 to xylose yeast yeast from sugarlignin Lignin Caustic insol Sugar extract ferm yeast prop slurryinoculum extraction extraction extract fibers 12 13 14 15 16 17 18 19 20Component & Units 116.2 0.31 Total Flow kg/hr 118.49 14.40 0.03 8.2984.45 68.36 132.30 57.05 Dry total solids, kg/h 14.69 0.12 0.10 0.3727.34 0.68 6.78 19.97 Total Solids, wt fraction 0.12 0.88 0.90 0.04 0.320.01 0.05 0.35 Moisture, wt fraction 0.88 0.00 0.10 0.96 0.68 0.99 0.950.65 Insoluble, wt fraction 0.00 0.12 0.00 0.01 0.32 0.00 0.00 0.35Soluble solids, wt fraction 0.12 0.26 0.03 0.03 0.01 0.01 0.04 0.00Insoluble, kg/hr 0.27 14.13 0.00 0.10 26.58 0.00 0.27 19.95 Solublesolids, kg/hr 14.42 32.0 40.0 0.27 0.76 0.68 5.70 0.01 Temperature ° C.65.0 5.00 6.80 65.0 45.0 45.0 45.0 pH 2.50 3.70 13.00 12.00 8.00Pressure, psig Steam, kg/hr Water kg/hr 103.80 101.72 0.27 7.27 58.1067.67 125.78 37.08 Ethanol kg/hr Glucose + oligomers (SS) kg/hr 1.701.66 0.03 0.09 0.14 Xylose + oligomers (SS) kg/hr 8.76 8.59 0.18 0.460.18 Arabinose + oligomers (SS) kg/hr 1.23 1.21 0.02 0.06 0.06Non-glucose C6 Sugar + 1.04 1.02 0.02 0.05 0.05 oligomers (SS) kg/hrInorganic Salts (SS) kg/hr 0.36 0.35 0.01 0.02 0.02 Volatile organics(acetic acid + 0.96 0.95 0.02 0.05 0.05 furfural + HMF) kg/hr LacticAcid kg/hr Uronic Acid kg/hr 0.61 0.60 0.01 0.03 0.03 Ammonia (NH3)kg/hr NaOH kg/h 0.68 Sulfuric acid kg/hr 0.89 0.06 0.05 0.67 0.01 CarbonDioxide kg/hr 0.05 Oxygen kg/hr Nitrogen kg/hr Starch (IS) kg/hr Glucan(IS) kg/hr 14.47 14.32 Xylan (IS) kg/hr 0.90 0.76 Arabinan (IS) kg/hr0.30 0.24 Non-glucose C6 Solid (IS) kg/hr 0.23 0.18 Lignin (IS) kg/hr7.52 2.26 Acetate (IS) kg/hr 0.04 0.04 Uronic Acid (IS) kg/hr 0.64 0.64Ash (IS) kg/hr 1.50 1.50 Protein kg/hr 0.03 Yeast (IS) kg/h 0.10 Enzyme(IS) kg/hr Unknown (IS) kg/hr Unknown (SS) kg/hr Corn mash (35% TS),kg/hr 75% glucose syrup Nutrients, kg/hr Caustic insol Caustic insol Enzpdn Crude Water to Enz C6 yeast C6 dry C6 yeast fibers to enz pdn fibersto hyd supplement enzyme Enz Hyd Hydrolysate suppl yeast inoculum 21 2223 24 25 26 27 28 29 Component & Units Total Flow kg/hr 0.57 56.48 2.592.81 42.36 112.21 5.61 Dry total solids, kg/h 0.20 19.77 0.21 29.06 0.010.12 Total Solids, wt fraction 0.35 0.35 0.07 0.26 0.02 Moisture, wtfraction 0.65 0.65 0.93 0.74 Insoluble, wt fraction 0.35 0.35 0.07 0.13Soluble solids, wt fraction 0.00 0.00 0.13 Insoluble, kg/hr 0.20 19.7714.26 Soluble solids, kg/hr 0.00 0.01 14.60 Ethanol Concentration, wt %Temperature ° C. 45 45 35 35 45 45 pH 8.0 8.0 5.0 5.0 Pressure, psigSteam Water kg/hr 36.71 1.49 2.59 42.36 83.15 Ethanol kg/hr 5.49 Glucose(SS) kg/hr 0.35 13.51 Xylose (SS) kg/hr 0.81 Arabinose (SS) kg/hr 0.27C6 Sugar (SS) kg/hr 0.20 Lignin (SS) kg/hr Inorganic Salts (SS) kg/hrVolatile organics (acetic acid + furfural + HMF) kg/hr Lactic Acid kg/hrUronic Acid kg/hr Ammonia (NH3) kg/hr NaOH kg/h Sulfuric acid kg/hrCarbon Dioxide kg/hr Oxygen kg/hr Nitrogen kg/hr Starch (IS) kg/hrGlucan (IS) kg/hr 14.32 2.15 Xylan (IS) kg/hr 0.89 0.18 Arabinan (IS)kg/hr 0.30 0.30 C6 Solid (IS) kg/hr 0.23 0.23 Lignin (IS) kg/hr 7.457.45 Acetate (IS) kg/hr 0.04 0.04 Uronic Acid (IS) kg/hr 0.64 0.64 Ash(IS) kg/hr 1.49 1.49 Protein kg/hr 0.21 1.71 Yeast (IS) kg/h 0.01 0.08Enzyme (IS) kg/hr Unknown (IS) kg/hr Unknown (SS) kg/hr Corn mash (35%TS), kg/hr 75% glucose syrup 0.47 0.37 Nutrients, kg/hr 0.27 0.08 0.0493% sulfuric acid Lignin Distillation Whole Solid to lignin powder C5beer C6 beer feed High-wine stillage residue Thin Stillage precipitateproduct 30 31 32 33 34 35 36 37 38 Component & Units Total Flow kg/hr119.88 117.82 237.71 25.12 243.58 52.88 190.64 1.06 5.27 Dry totalsolids, kg/h 5.04 16.82 21.86 20.57 18.51 2.06 4.74 Total Solids, wtfraction 0.04 0.14 0.09 0.08 0.35 0.01 0.90 Moisture, wt fractionInsoluble, wt fraction Soluble solids, wt fraction Insoluble, kg/hrSoluble solids, kg/hr Ethanol Concentration, wt % 3.80 6.63 0.05 42.000.00 Temperature ° C. 35 37 36 50 90 pH 4.5 4.5 4.5 4.5 Pressure, psigSteam Water kg/hr 108.99 88.64 197.63 14.57 222.96 34.37 188.58 0.07Ethanol kg/hr 4.31 6.29 10.60 10.55 0.05 Glucose (SS) kg/hr 0.42 1.351.77 1.77 Xylose (SS) kg/hr 2.15 0.81 2.96 2.96 Arabinose (SS) kg/hr1.21 0.27 1.48 1.48 C6 Sugar (SS) kg/hr 0.25 0.02 Lignin (SS) kg/hr 0.45Inorganic Salts (SS) kg/hr 0.35 Volatile organics (acetic acid +furfural + 0.95 HMF) kg/hr Lactic Acid kg/hr Uronic Acid kg/hr Ammonia(NH3) kg/hr 0.60 NaOH kg/h Sulfuric acid kg/hr 0.98 Carbon Dioxide kg/hrOxygen kg/hr Nitrogen kg/hr Starch (IS) kg/hr Glucan (IS) kg/hr 2.152.15 Xylan (IS) kg/hr 0.18 0.18 Arabinan (IS) kg/hr 0.30 0.30 C6 Solid(IS) kg/hr 0.23 0.23 Lignin (IS) kg/hr 7.45 7.45 4.74 Acetate (IS) kg/hr0.04 0.04 Uronic Acid (IS) kg/hr 0.64 0.64 Ash (IS) kg/hr 1.49 1.49Protein kg/hr 1.71 1.71 Yeast (IS) kg/h 0.22 0.18 0.18 Enzyme (IS) kg/hrUnknown (IS) kg/hr Unknown (SS) kg/hr Corn mash (35% TS), kg/hr 75%glucose syrup Nutrients, kg/hr Lignin filtrate Lignin slurry product(alternate) Distillation steam 39 40 41 Component & Units Total Flowkg/hr 152.13 17.56 39.90 Dry total solids, kg/h 2.83 5.27 Total Solids,wt fraction 0.02 0.30 Moisture, wt fraction Insoluble, wt fractionSoluble solids, wt fraction Insoluble, kg/hr Soluble solids, kg/hrEthanol Concentration, wt % Temperature ° C. 160 pH Pressure, psig 75.0Steam 39.90 Water kg/hr Ethanol kg/hr Glucose (SS) kg/hr Xylose (SS)kg/hr Arabinose (SS) kg/hr C6 Sugar (SS) kg/hr Lignin (SS) kg/hrInorganic Salts (SS) kg/hr Volatile organics (acetic acid + furfural +HMF) kg/hr Lactic Acid kg/hr Uronic Acid kg/hr Ammonia (NH3) kg/hr NaOHkg/h Sulfuric acid kg/hr Carbon Dioxide kg/hr Oxygen kg/hr Nitrogenkg/hr Starch (IS) kg/hr Glucan (IS) kg/hr Xylan (IS) kg/hr Arabinan (IS)kg/hr C6 Solid (IS) kg/hr Lignin (IS) kg/hr Acetate (IS) kg/hr UronicAcid (IS) kg/hr Ash (IS) kg/hr Protein kg/hr Yeast (IS) kg/h Enzyme (IS)kg/hr Unknown (IS) kg/hr Unknown (SS) kg/hr Corn mash (35% TS), kg/hr75% glucose syrup Nutrients, kg/hr

EXAMPLE 6

This example provides a mass balance (Table 11) for an ethanolproduction process of the present invention prepared using corn stoverand generally corresponding to the process depicted in FIG. 3.

TABLE 11 Milled & Pretreatment Conditioned cleaned Dilute AcidifiedPretreatment flash PCS CS Acid CS steam steam PCS Reactants slurryEnzyme Component & Units 1 2 3 4 5 6 7 8 9 Total Flow kg/hr 46.30 48.3894.68 27.46 13.61 106.06 16.17 122.23 0.95 Dry total solids, kg/h 41.6740.42 1.62 42.03 0.07 Total Solids, wt fraction 0.90 0.38 0.10 0.34 0.08Moisture, wt fraction 0.10 0.62 0.90 0.66 0.92 Insoluble, wt fraction1.00 0.65 0.36 0.64 Soluble solids, wt fraction 0.00 0.35 0.54 0.36Insoluble, kg/hr 26.27 0.58 26.85 0.07 Soluble solids, kg/hr 14.15 1.0315.18 Temperature ° C. 20 60 60 192 100 50 60 20 pH 1.1 1.8 4.5 4.5 4.5Pressure, psig 14.7 175.0 Steam, kg/hr 27.46 13.61 Water kg/hr 4.6347.44 52.07 65.64 14.55 80.19 0.88 Ethanol kg/hr Glucose + oligomers(SS) kg/hr 1.78 Xylose + oligomers (SS) kg/hr 9.22 Arabinose + oligomers(SS) kg/hr 1.30 Non-glucose C6 Sugar + oligomers (SS) 1.10 kg/hr Lignin(SS) kg/hr 0.48 Inorganic Salts (SS) kg/hr 0.38 Volatile organics(acetic acid + furfural + 1.02 HMF) kg/hr Lactic Acid kg/hr Uronic Acidkg/hr 0.64 Ammonia (NH3) kg/hr NaOH kg/h Sulfuric acid kg/hr 0.94 0.940.94 Carbon Dioxide kg/hr Oxygen kg/hr Nitrogen kg/hr Starch (IS) kg/hrGlucan (IS) kg/hr 16.08 16.08 14.47 Xylan (IS) kg/hr 9.02 9.02 0.90Arabinan (IS) kg/hr 1.45 1.45 0.30 Non-glucose C6 Solid (IS) kg/hr 1.221.22 0.23 Lignin (IS) kg/hr 8.00 8.00 7.52 Acetate (IS) kg/hr 0.83 0.830.04 Uronic Acid (IS) kg/hr 1.29 1.29 0.64 Ash (IS) kg/hr 1.88 1.88 1.50Protein kg/hr 1.50 1.50 0.07 Yeast (IS) kg/h Enzyme (IS) kg/hr Unknown(IS) kg/hr 0.40 0.40 0.40 Unknown (SS) kg/hr Corn mash (35% TS), kg/hr75% glucose syrup Nutrients, kg/hr PCS Sugar extract Suplement XyloseXylose Washed fibers Washed fibers feed to sugar Wash Sugar to xylose 1to xylose yeast yeast from sugar to enz extraction water extract fermyeast prop slurry inoculum extraction production Component & Units 10 1112 13 14 15 16 17 18 Total Flow kg/hr 123.18 80.83 118.49 116.12 0.318.29 85.45 0.85 Dry total solids, kg/h 42.11 14.69 14.40 0.03 0.37 27.340.27 Total Solids, wt fraction 0.34 0.12 0.12 0.10 0.04 0.32 0.32Moisture, wt fraction 0.66 0.88 0.88 0.90 0.96 0.68 0.68 Insoluble, wtfraction 0.22 0.00 0.00 0.10 0.01 0.31 0.32 Soluble solids, wt fraction0.12 0.12 0.12 0.00 0.03 0.01 0.00 Insoluble, kg/hr 26.93 0.27 0.26 0.030.10 26.58 0.27 Soluble solids, kg/hr 15.18 14.42 14.13 0.00 0.27 0.760.00 Temperature ° C. 60 70 65 32 40 65 45 pH 4.5 6.8 2.5 5.0 6.8 3.73.7 Pressure, psig Steam, kg/hr Water kg/hr 81.07 80.83 103.80 101.720.27 7.27 58.10 0.58 Ethanol kg/hr Glucose + oligomers (SS) kg/hr 1.701.66 0.03 Xylose + oligomers (SS) kg/hr 8.76 8.59 0.18 Arabinose +oligomers (SS) kg/hr 1.23 1.21 0.02 Non-glucose C6 Sugar + oligomers1.04 1.02 0.02 (SS) kg/hr Lignin (SS) kg/hr 0.46 0.45 0.01 InorganicSalts (SS) kg/hr 0.36 0.35 0.01 Volatile organics (acetic acid + 0.960.95 0.02 furfural + HMF) kg/hr Lactic Acid kg/hr Uronic Acid kg/hr 0.610.60 0.01 Ammonia (NH3) kg/hr NaOH kg/h Sulfuric acid kg/hr 0.89 0.06Carbon Dioxide kg/hr Oxygen kg/hr Nitrogen kg/hr Starch (IS) kg/hr 14.470.14 Glucan (IS) kg/hr 0.90 0.01 Xylan (IS) kg/hr 0.30 0.00 Arabinan(IS) kg/hr 0.23 0.00 Non-glucose C6 Solid (IS) kg/hr 7.52 0.08 Lignin(IS) kg/hr 0.04 0.00 Acetate (IS) kg/hr 0.64 0.01 Uronic Acid (IS) kg/hr1.50 0.02 Ash (IS) kg/hr Protein kg/hr 0.10 Yeast (IS) kg/h 0.03 Enzyme(IS) kg/hr Unknown (IS) kg/hr Unknown (SS) kg/hr Corn mash (35% TS),kg/hr 75% glucose syrup Nutrients, kg/hr Washed fibers to enz hydrolysisEnz pdn supplement Component & Units 19 20 Total Flow kg/hr 84.59 2.59Dry total solids, kg/h 27.07 Total Solids, wt fraction 0.32 Moisture, wtfraction 0.68 Insoluble, wt fraction 0.31 Soluble solids, wt fraction0.01 Insoluble, kg/hr 26.31 Soluble solids, kg/hr 0.76 Temperature ° C.45 35 pH 3.7 5.0 Pressure, psig Steam, kg/hr Water kg/hr 58.69 1.49Ethanol kg/hr Glucose + oligomers (SS) kg/hr 0.09 0.35 Xylose +oligomers (SS) kg/hr 0.46 Arabinose + oligomers (SS) kg/hr 0.06Non-glucose C6 Sugar + oligomers (SS) kg/hr 0.05 Lignin (SS) kg/hr 0.02Inorganic Salts (SS) kg/hr 0.02 Volatile organics (acetic acid +furfural + 0.05 HMF) kg/hr Lactic Acid kg/hr Uronic Acid kg/hr 0.03Ammonia (NH3) kg/hr NaOH kg/h Sulfuric acid kg/hr 0.05 Carbon Dioxidekg/hr Oxygen kg/hr Nitrogen kg/hr 14.32 Starch (IS) kg/hr 0.89 Glucan(IS) kg/hr 0.30 Xylan (IS) kg/hr 0.23 Arabinan (IS) kg/hr 7.45Non-glucose C6 Solid (IS) kg/hr 0.04 Lignin (IS) kg/hr 0.64 Acetate (IS)kg/hr 1.49 Uronic Acid (IS) kg/hr Ash (IS) kg/hr Protein kg/hr Yeast(IS) kg/h Enzyme (IS) kg/hr Unknown (IS) kg/hr Unknown (SS) kg/hr Cornmash (35% TS), kg/hr 75% glucose syrup 0.47 Nutrients, kg/hr 0.27 CrudeWater to enz Enzyme C6 yeast C6 dry C6 yeast Distillation enzymehydrolysis Hydrolysate suppl yeast inoculum C5 beer C6 beer feedComponent & Units 21 22 23 24 25 26 27 28 29 Total Flow kg/hr 2.81 50.76141.28 2.81 0.00 7.06 119.88 148.34 268.23 Dry total solids, kg/h 0.2129.25 0.21 0.07 1.20 5.04 17.76 22.80 Total Solids, wt fraction 0.070.21 0.07 0.17 0.04 0.12 0.08 Moisture, wt fraction 0.93 0.79 0.93 0.830.96 0.88 0.92 Insoluble, wt fraction 0.07 0.10 0.07 0.17 0.00 0.11 0.06Soluble solids, wt fraction 0.10 0.00 0.02 Insoluble, kg/hr 14.45 1.200.22 16.65 16.87 Soluble solids, kg/hr 14.60 0.00 4.82 1.10 5.93 EthanolConcentration, wt % 3.80 5.07 4.47 Temperature ° C. 35 45 45 35 45 45 3535 35 pH 5.0 7.0 4.5 5.0 4.5 4.5 4.5 4.5 Pressure, psig Steam Waterkg/hr 2.59 50.76 112.03 2.59 5.86 108.99 117.89 226.88 Ethanol kg/hr4.31 6.29 10.60 Glucose (SS) kg/hr 13.51 0.42 1.35 1.77 Xylose (SS)kg/hr 0.81 2.15 0.81 2.96 Arabinose (SS) kg/hr 0.27 1.21 0.27 1.48 C6Sugar (SS) kg/hr 0.20 0.25 0.02 0.27 Lignin (SS) kg/hr 0.45 0.45Inorganic Salts (SS) kg/hr 0.35 0.35 Volatile organics (acetic acid +furfural + 0.95 0.95 HMF) kg/hr Lactic Acid kg/hr Uronic Acid kg/hr 0.600.60 Ammonia (NH3) kg/hr NaOH kg/h Sulfuric acid kg/hr Carbon Dioxidekg/hr Oxygen kg/hr Nitrogen kg/hr Starch (IS) kg/hr Glucan (IS) kg/hr2.15 2.15 2.15 Xylan (IS) kg/hr 0.18 0.18 0.18 Arabinan (IS) kg/hr 0.300.30 0.30 C6 Solid (IS) kg/hr 0.23 0.23 0.23 Lignin (IS) kg/hr 7.45 7.457.45 Acetate (IS) kg/hr 0.04 0.04 0.04 Uronic Acid (IS) kg/hr 0.64 0.640.64 Ash (IS) kg/hr 1.49 1.49 1.49 Protein kg/hr 0.21 1.71 0.21 1.711.71 Yeast (IS) kg/h 0.07 1.12 0.22 1.12 1.12 Enzyme (IS) kg/hr Unknown(IS) kg/hr Unknown (SS) kg/hr Corn mash (35% TS), kg/hr 75% glucosesyrup Nutrients, kg/hr 0.27 0.08 .56 Caustic to 93% sulfuric LigninWhole Stillage Thin lignin Lignin acid to lignin powder Lignin filtrateto stillage High-wine cake stillage extraction extract precipitateproduct wastewater Component & Units 30 31 32 33 34 35 36 37 38 TotalFlow kg/hr 280.19 25.12 305.31 223.16 128.55 154.46 1.20 7.30 177.38 Drytotal solids, kg/h 22.80 16.95 5.85 0.80 8.74 6.94 2.39 Total Solids, wtfraction 0.08 0.06 0.03 0.01 0.06 0.95 0.01 Moisture, wt fraction 0.920.94 0.97 0.05 Insoluble, wt fraction 0.06 0.95 0.14 0.98 Solublesolids, wt fraction 0.02 0.05 0.86 0.02 Insoluble, kg/hr 16.87 16.030.84 6.83 Soluble solids, kg/hr 5.93 0.92 5.00 8.74 0.11 EthanolConcentration, wt % 0.02 42.00 0.02 0.02 Temperature ° C. 35 50 90 90 2050 25 pH 4.5 4.5 4.5 13.0 12.0 3.0 Pressure, psig Steam Water kg/hr257.34 14.57 40.07 217.32 127.75 145.72 0.08 174.98 Ethanol kg/hr 0.0510.55 0.01 0.04 Glucose (SS) kg/hr 1.77 Xylose (SS) kg/hr 2.15 Arabinose(SS) kg/hr 0.30 C6 Sugar (SS) kg/hr 0.25 0.79 Lignin (SS) kg/hr 0.116.33 0.32 Inorganic Salts (SS) kg/hr 0.09 1.28 Volatile organics (aceticacid + furfural + 0.24 HMF) kg/hr Lactic Acid kg/hr Uronic Acid kg/hr0.15 Ammonia (NH3) kg/hr 0.80 0.76 1.12 NaOH kg/h Sulfuric acid kg/hrCarbon Dioxide kg/hr Oxygen kg/hr Nitrogen kg/hr Starch (IS) kg/hrGlucan (IS) kg/hr 2.15 0.86 Xylan (IS) kg/hr 0.18 Arabinan (IS) kg/hr0.30 C6 Solid (IS) kg/hr 0.23 Lignin (IS) kg/hr 7.45 6.33 Acetate (IS)kg/hr 0.04 Uronic Acid (IS) kg/hr 0.64 Ash (IS) kg/hr 1.49 Protein kg/hr1.71 Yeast (IS) kg/h 1.12 Enzyme (IS) kg/hr Unknown (IS) kg/hr Unknown(SS) kg/hr Corn mash (35% TS), kg/hr 75% glucose syrup Nutrients, kg/hrLignin slurry product (alternate) Residue Distillation steam Component &Units 39 40 41 Total Flow kg/hr 29.13 31.11 45.02 Dry total solids, kg/h8.74 9.01 Total Solids, wt fraction 0.30 0.30 Moisture, wt fractionInsoluble, wt fraction Soluble solids, wt fraction Insoluble, kg/hr 8.84Soluble solids, kg/hr 8.74 0.13 Ethanol Concentration, wt % Temperature° C. 160 pH 12.0 Pressure, psig 75.0 Steam 45.02 Water kg/hr Ethanolkg/hr Glucose (SS) kg/hr Xylose (SS) kg/hr Arabinose (SS) kg/hr C6 Sugar(SS) kg/hr Lignin (SS) kg/hr Inorganic Salts (SS) kg/hr Volatileorganics (acetic acid + furfural + HMF) kg/hr Lactic Acid kg/hr UronicAcid kg/hr Ammonia (NH3) kg/hr NaOH kg/h 0.04 Sulfuric acid kg/hr CarbonDioxide kg/hr Oxygen kg/hr Nitrogen kg/hr Starch (IS) kg/hr Glucan (IS)kg/hr Xylan (IS) kg/hr Arabinan (IS) kg/hr C6 Solid (IS) kg/hr Lignin(IS) kg/hr Acetate (IS) kg/hr Uronic Acid (IS) kg/hr Ash (IS) kg/hrProtein kg/hr Yeast (IS) kg/h Enzyme (IS) kg/hr Unknown (IS) kg/hrUnknown (SS) kg/hr Corn mash (35% TS), kg/hr 75% glucose syrupNutrients, kg/hr

EXAMPLE 7

This example demonstrates the relationship between particle size and ashcontent of milled dried corn stover.

A 30 g sample of 1.25 inch roto-chopped corn stover was dried in aconvection oven at 45° C. under atmospheric pressure (760 mm Hgabsolute; 101.3 kPa) over 20 hours. After 20 hours, the chopped cornstover was weighed.

Then the chopped corn stover was milled in a kinematic knife millequipped with a 2 mm outlet screen. The milled corn stover particlespassing through the 2 mm screen were classified in a Tyler sieve shakercontaining a 20 Mesh screen (840 μm) and a 100 Mesh screen (150 μm).

Approximately 2.0 g samples of each corn stover fraction (i.e., the1.25-inch roto-chopped, 2 mm, >20 mesh, >100 mesh to <20 mesh, and <100mesh fraction) were placed in a crucible, and heated in a muffle furnaceequipped with a thermostat, set to 575 (±25° C.) for 22 hours. After 22hours the weight of each sample was measured, which represents the totalash content of the dried corn stover samples.

Table 12 shows the total ash content of the dried corn stover fractions.The results show that the <100 Mesh fraction has over 80 wt % ash andthe >20 Mesh fraction has the lowest percentage of ash at about 6.5 wt%.

TABLE 12 Experiment 1 Experiment 2 Average Sample (wt %) (wt %) (wt %)1.25-inch roto-chopped 7.91 9.60 8.75 2 mm 17.51 16.60 17.05 >20 mesh6.55 5.86 6.21 >100 mesh to <20 mesh 24.77 25.63 25.20 <100 mesh 81.9881.55 81.76

EXAMPLE 8

The example describes Protocol A for determining the acid neutralizationcapacity of a biomass feedstock and fractions thereof (e.g., fineparticulate fractions and cleaned biomass feedstocks).

Step 1: Determine the Dry Weight (W_(s)) of the Biomass Sample.

To determine the dry weight of the biomass sample, place a 30 g samplein a convection oven at 105° C. under atmospheric pressure (760 mm Hgabsolute; 101.3 kPa) until constant weight is achieved (i.e., change inweight is less than +/−1 wt % upon reheating) and then weigh the sample.

Step 2: Determine pH_(initial) of a Standard Acid Sulfuric AcidSolution.

In a 1000 ml beaker place 500 ml of a standard sulfuric acid solution(e.g., 0.01N, 0.02N, or 0.05N solution). Measure the pH value of thestandard sulfuric acid solution using a calibrated pH meter.

Step 3: Determine pH_(final) of the Slurry

Add the 30 g (dry weight) of the biomass sample to the standard sulfuricacid solution in the 1000 ml beaker. Place the beaker with a magneticagitator in a water bath at 25° C., and stir at 330 revolutions perminute (rpm) for 30 minutes. After the 30 minutes, measure the pH valueof the slurry with the calibrated pH meter.

Step 4: Calculate the Acid Neutralizing Capacity

Acid neutralizing capacity (ANC: g/g) of the sample is calculatedaccording to the following equation:ANC=[H⁺](neutralized)×V×MW×MR×1/W_(s)

-   -   wherein,    -   [H⁺](neutralized)=(10^(−pHinitial)−10^(−pHfinal)) (mol/L)    -   V=total volume of slurry (L)    -   MW=molecular weight of sulfuric acid (i.e., 98 g/mol)    -   MR=mole ratio of sulfuric acid to hydrogen ion (i.e., ½)    -   W_(s)=dry weight of sample (g)

EXAMPLE 9

This example compares the acid neutralization capacity as determined inaccordance with Protocol A for three corn stover samples, including theeffects of screening the milled corn stover using a screen havingopenings of a size of about U.S. Sieve No. 60 (250 μm). Table 13provides the ash content and acid neutralization capacity for thefractions tested.

TABLE 13 Dry, Milled Corn Stover Sample 1 Sample 2 Sample 3 ApproximateWeight (g) 100 100 100 Ash content (wt %) 5.4 9.0 11.0 AcidNeutralization Capacity 0.0054 0.0108 0.0148 (g sulfuric acid/g drymatter) Screened & Milled Corn Stover (g) 96.6 95.4 93.9 Ash Content ofScreened & 4.0 7.0 7.8 Milled Corn Stover (wt %) Acid NeutralizationCapacity of 0.0038 0.0080 0.0107 Screened & Milled Corn Stover (gsulfuric acid/ g dry matter) Fines (g) 3.4 4.7 6.1 Ash Content of Fines(wt %) 45.0 50.0 60.0 Acid Neutralization Capacity of 0.0093 0.01400.0206 Fines (g sulfuric acid/ g dry matter)

EXAMPLE 10

Protocol B for determining the xylose content of a biomass feedstock,pretreated biomass feedstock, and fractions thereof, which is necessaryfor determining xylose yield (based on the hemicellulose content of thebiomass feedstock) is the method described in the National RenewableEnergy Laboratory (NREL) Technical Report NREL/TP-510-42623, January2008, which is entitled “Determination of Sugars, Byproducts, andDegradation Products in Liquid Fraction Process Samples,” LaboratoryAnalytical Procedure (LAP), Issue Date: Dec. 8, 2006, by A. Sluiter, B.Hames, R. Ruiz, C. Scarlata, J. Sluiter, and D. Templeton. The entirecontents of this report are hereby incorporated herein by reference forall relevant purposes.

EXAMPLE 11

This example describes Protocol C for determining the cellulosedigestibility of a biomass feedstock, pretreated biomass feedstock, andfractions thereof.

This protocol describes the enzymatic saccharification of cellulose fromnative or pretreated lignocellulosic biomass to glucose in order todetermine the maximum extent of digestibility possible. This protocolcovers the determination of the maximum extent of digestibility oflignocellulosic biomass. If the biomass is suspected to have some starchcontent, the dry weight percent cellulose calculated from total glucanmust be corrected to subtract the starch contribution to total dryweight percent glucose.

Samples should be washed to remove any free acid or alkali prior toconducting this protocol.

1. Sampling and Test Specimens

The test specimen consists of about 1 gram of 6 wt % total solidspretreated biomass sample obtained in such a manner as to ensure that itis representative of the entire lot of material being tested.

All lignocellulosic materials which have undergone some aqueouspretreatment must not have undergone any drying prior to enzymedigestibility, since irreversible pore collapse can occur in themicro-structure of the biomass leading to decreased enzymatic release ofglucose from the cellulose. Additionally, all frozen lignocellulosicmaterials which are to be subjected to digestibility tests can not havebeen frozen for more than one month prior to analysis, since, dependingon the environment, sublimation could have occurred leading to possibleirreversible collapse of micro-pores in the biomass.

2. Apparatus & Materials

-   -   Incubator set at 50±1° C.    -   Micro-centrifuge    -   pH meter    -   Analytical balance: sensitive to 0.0001 grams.    -   HPLC column with refractive index detector and BioRad Aminex®        HPX-87P column    -   Drying oven adjusted to 105±2° C.    -   A 10, 20, 200 μL and a 1000 μL pipetteman with corresponding        tips    -   A pipette tip clipper for sampling high solids slurries    -   250 ml or 500 mL baffled glass shake flasks equipped with        plastic-lined caps or rubber stoppers or drilled rubber stoppers        fitted with airlocks or bubble traps

3. Reagents

a) Cellulase enzyme (e.g., Celluclast 1.5 L from Novozymes, Accellerase1000 from Genencor, and 22 CG from Novozymes) of known activity (e.g.,FPU/mL). In some cases, a different unit of activity may be specified bythe enzyme manufacturer so that the enzyme loadings can be compared on aweight and/or cost basis; b) Sodium citrate buffer (1M, ph 4.8); c)β-glucosidase enzyme of known activity, p-nitrophenyl-glucoside units(pNPGU/mL) (This is only for Celluclast 1.5 L); d) Distilled water orreverse osmosis purified water; e) LACTROL

4. Sample Preparation

Determine the Total Solids (TS %/100) for all cellulose containingsamples to be digested.

Weigh out about 1 gram of 6 wt % total solids pretreated biomass sampleand add to a 250 mL flask. To each flask, add 5.0 mL 1.0 M, pH 4.8sodium citrate buffer.

To each flask, add 0.5mg LACTROL to prevent the growth of organismsduring the digestion.

Calculate the amount of distilled water needed to bring the total volumein each flask to 100.00 mL after addition of the enzymes specified inthe following step. Add the appropriate calculated volume of water toeach flask. All solutions and the biomass are assumed to have a specificgravity of 1.000 g/mL. Thus, if 6.0 g of biomass is added to the flask,it is assumed to occupy 6.0 mL and 94 mL of total liquid is to be added.

Bring the contents of each flask to 50° C. by warming in the incubatorset at 50±1° C. To each flask is added an appropriate volume of thecellulase enzyme preparation to equal 5 FPU/g glucan (for Celluclast 1.5L) and the appropriate volume of β-glucosidase enzyme to equal 7.5p-nitrophenyl-glucoside units (pNPGU)/g glucan for a ratio of 1:1.5 ofcellulase to glucosidase. This ratio may not be possible if the twoenzymes come premixed in a cocktail so the enzyme loading should beadjusted accordingly. The rate of enzymatic release of glucose is to bemeasured; all contents of the container prior to the addition of theenzyme must be at 50° C. and pH=4.8. Enzymes are added last since thereaction is initiated by the addition of enzyme.

Prepare a reaction blank for the substrate. The substrate blank containsbuffer, water, and the identical amount of substrate in a 100.00 mLvolume.

Prepare enzyme blanks for cellulase and β-glucosidase with buffer,water, and the identical amount of the enzyme.

Close the vials tightly and incubate with gentle rotation (150 RPM) fora period of 72 to 110 hours or until the release of soluble sugars fromthe sample(s) becomes negligible.

If the progress of the reaction is to be measured, a 0.3-0.5 mL aliquotis removed at each predetermined time interval (0, 6, 12, 24, 48, 72, 96hrs) after the vial contents have been well mixed by shaking. The sampleis heated at 100° C. for 10 minutes to inactivate the enzyme, the sampleis the cooled down and expelled into a 1.5 mL microcentrifuge tube andcentrifuged for 5 minutes. The supernatant is subjected to glucoseanalysis using HPLC.

5. Calculations

To calculate the percent digestibility of the cellulose added to thecontainer, determine glucose concentration in the centrifugedsupernatant by HPLC. Subtract the glucose concentrations, if any, fromthe substrates and enzyme blanks. Correct for hydration by multiplyingthe glucose reading by 0.9 to correct for the water molecule added uponhydrolysis of the cellulose polymer and multiply by 100 mL total volumeof assay.

Example: If the glucose analyzer reading (corrected with blanks) is 9.9mg/mL, then the amount of glucan digested is: 0.0099 g/mL×100 mL×0.9=0.891 g

Calculate Cellulose Digestibility:Cellulose Digestibility(%)=100%×(Gram Cellulose Digested/Grams CelluloseAdded)

6. Notes and Precautions

Report the results to two decimal places, on a 105° C. dry basis. Forreplicate analyses of the same sample, report the average, standarddeviation and relative percent difference (% RPD).

b) Relative percent difference criteria: Not defined; depend on thesubstrate being tested. Different preparations of pretreated biomasswill exhibit different amount of homogeneity, which will influence theextent to which they are hydrolyzed.

Sample storage: Store pretreated samples should be stored moist, frozenin a well sealed container or vacuum packed not longer than threemonths.

The present invention is not limited to the above embodiments and can bevariously modified. The above description of the preferred embodiments,including the Examples, is intended only to acquaint others skilled inthe art with the invention, its principles, and its practicalapplication so that others skilled in the art may adapt and apply theinvention in its numerous forms, as may be best suited to therequirements of a particular use.

With reference to the use of the word(s) comprise or comprises orcomprising in this entire specification (including the claims below),unless the context requires otherwise, those words are used on the basisand clear understanding that they are to be interpreted inclusively,rather than exclusively, and applicants intend each of those words to beso interpreted in construing this entire specification.

When introducing elements of the present invention or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

What is claimed is:
 1. A method for pretreatment of virgin cellulosicbiomass feedstock comprising cellulose, hemicellulose, lignin, and sand,the method comprising: dry cleaning the virgin cellulosic biomassfeedstock in a fine particulate separation zone comprising at least oneclassifying screen having openings of from U.S. Sieve No. 20 (840 μm) toU.S. Sieve No. 100 (150 μm) to recover a cleaned biomass feedstock onthe at least one classifying screen and remove a fine particulatefraction therefrom, the fine particulate fraction having passed throughthe classifying screen wherein the fine particulate fraction has an ashcontent of at least about 30 wt. %, wherein said dry cleaning is done inthe absence of the addition of liquid to the virgin cellulosic biomassfeedstock for the purpose of removing contaminants; contacting in anacid impregnation vessel the cleaned biomass feedstock and an acidicaqueous liquid medium containing less than 5 wt % acid to form anacid-impregnated cellulosic biomass feedstock; removing an aqueousliquid fraction from the acid-impregnated cellulosic biomass feedstockto form an acid-impregnated feedstock having a reduced sand content andan acidic aqueous liquid fraction comprising sand; removing a sand-richproduct from the acidic aqueous liquid fraction; and introducing atleast a portion of the acidic aqueous liquid fraction having thesand-rich product removed therefrom into the acid impregnation vessel.2. The method of claim 1 wherein the cleaned biomass feedstock and theacidic aqueous liquid medium are contacted by soaking the cleanedbiomass feedstock in the acidic liquid medium.
 3. The method of claim 1wherein the acidic aqueous liquid fraction having the sand rich productremoved therefrom and the acidic aqueous liquid medium are combinedprior to introduction into the acid-impregnation vessel.
 4. The methodof claim 1 wherein the cellulosic biomass feedstock comprises particleshaving a particle size distribution such that at least 50 wt % of thefeedstock particles have a size in their largest dimension of from about0.6 cm (0.25 inches) to about 4 cm (1.5 inches).
 5. The method of claim1 wherein the acidic aqueous liquid medium includes the acid at aconcentration of from about 0.7 wt % to about 3.5 wt %.
 6. The method ofclaim 1 wherein the acidic aqueous liquid medium includes the acid at aconcentration of from about 1.0 wt % to about 3.0 wt %.
 7. The method ofclaim 1 wherein the cleaned biomass feedstock is contacted with at least0.005 kg acid (acid weight basis) per kg cellulosic biomass feedstock(dry weight basis).
 8. The method of claim 1 wherein the cleaned biomassfeedstock is contacted with from about 0.01 kg to about 0.05 kg acid(acid weight basis) per kg cellulosic biomass feedstock (dry weightbasis).
 9. The method of claim 1 wherein the acidic aqueous liquidmedium comprises a surfactant (wetting agent).
 10. The method of claim 1wherein the temperature of the acidic aqueous liquid medium is fromabout 20° C. to about 95° C.
 11. The method of claim 1 furthercomprising heating during contact of the cleaned biomass feedstock withthe acidic aqueous liquid medium.
 12. The method of claim 1 wherein thecleaned biomass feedstock and acidic aqueous liquid medium are contactedat a temperature of from about 30° C. to about 90° C.
 13. The method ofclaim 1 wherein the acid-impregnated cellulosic biomass feedstock has amoisture content of from about 20 wt % to about 70 wt %.
 14. The methodof claim 1 wherein the acid-impregnated cellulosic biomass feedstock hasa moisture content of from about 30 wt % to about 70 wt %.
 15. Themethod of claim 1 wherein the temperature of the acid impregnatedcellulosic biomass feedstock is from about 40° C. to about 80° C. 16.The method of claim 1 wherein the cleaned biomass feedstock is contactedwith the acidic aqueous liquid medium in the acid impregnation vesseland the acid-impregnated cellulosic biomass feedstock is removed fromthe acid impregnation vessel and held in a second vessel, the residencetime of the acid-impregnated cellulosic biomass feedstock in the secondvessel being from about 1 to about 60 minutes.
 17. The method of claim 1wherein the cleaned biomass feedstock is contacted with the acidicaqueous liquid medium by spraying the acidic liquid medium onto thecellulosic biomass feedstock.
 18. The method of claim 17 wherein thecleaned biomass feedstock is contacted with the acidic aqueous liquidmedium in an acid impregnation vessel comprising counter-rotating shaftshaving flights mounted thereon for agitation of the biomass.
 19. Themethod of claim 18 wherein the cleaned biomass feedstock is agitated forfrom about 2 to about 5 minutes.
 20. The method of claim 18 wherein thecleaned biomass feedstock is agitated at an intensity of from about 2kWh/ton biomass to about 10 kWh/ton biomass.
 21. The method of claim 18wherein the cleaned biomass feedstock is contacted with the acidicaqueous liquid medium in the acid impregnation vessel for from about 2to about 35 minutes.
 22. The method of claim 1 wherein the fineparticulate fraction has a moisture content of less than 20 wt. %. 23.The method of claim 1 wherein the cleaned biomass feedstock has amoisture content of less than 20 wt. %.
 24. The method of claim 1wherein the moisture content of the cleaned biomass feedstock and of thevirgin cellulosic biomass feedstock vary by no more than about 10 wt. %.25. The method of claim 1 wherein the moisture content of the cleanedbiomass feedstock and of the virgin cellulosic biomass feedstock vary byno more than about 5 wt. %.
 26. The method of claim 1 wherein themoisture content of the cleaned biomass feedstock and of the virgincellulosic biomass feedstock vary by no more than about 3 wt. %.